Download Interleukin 1, Interleukin 6, Tumor Necrosis

[CANCER RESEARCH 51, 2379-2385, May 1. 1991]
Inter leu kin 1, Interleukin 6, Tumor Necrosis Factor, and Transforming Growth
Factor ßIncrease Cell Resistance to Tumor Necrosis Factor Cytotoxicity by
Growth Arrest in the G! Phase of the Cell Cycle1
JoséE. Belizario and Charles A. Dinarello2
Department of Medicine, Tufts University School of Medicine, and New England Medical Center, Boston, Massachusetts 02111
ABSTRACT
During infection, inflammation, immune responses, and neoplastia
growth, various cytokines are produced affecting both susceptibility to
and protection from cellular death. We have studied the protective effect
of pretreatment of the L929 fibroblast cell line with interleukin 10 (II1/3), IL-6, tumor necrosis factor a (TNF-a), or transforming growth
factor 0, (TGF-/9) on subsequent TNF/actinomycin D-induced cytotoxicity. The protective effects of these cytokines on TNF cytotoxicity were
time and concentration dependent. TGF-/3 was the most effective cytokine,
followed by TNF, IL-1/3, and IL-6. Activators of protein kinase C also
afforded protection, and TGF-/3 acted synergistically with either phorbol
12-myristate 13-acetate or the calcium ionophore A-23187. TGF-/3induced protection against TNF was observed in cells subjected to pro
longed treatment with phorbol 12-myristate 13-acetate. Cells pretreated
with prostaglandin E2 or cholera toxin amplified the sensitivity to TNF
and inhibited TGF-/3-mediated resistance, whereas indomethacin en
hanced the protective effect of TGF-/3. Cells cultured in the presence of
IL-1/3, IL-6, TNF-a, or TGF-0 for 6 h inhibited DNA synthesis, and this
was associated with concomitant growth arrest in the Gì
phase of the cell
cycle. On the other hand, prostaglandin K; or cholera toxin stimulated
the progression of cells from G, toward G2 + M which was associated
with increased TNF sensitivity. We conclude that these cytokines protect
against death by arresting growth in the G, phase of the cell cycle.
INTRODUCTION
Cytokines are biologically potent polypeptides produced by a
variety of cells in response to infection, microbial toxins, in
flammatory agents, and neoplastic growth, as well as to them
selves. Several cytokines share the ability to stimulate cell
proliferation, initiate the synthesis of new proteins, and induce
the production of inflammatory metabolites in a wide variety
of cells. Of the various cytokines which have been identified,
considerable interest has focused on IL-13 (1), IL-6 (2), TNF
(3), TGF-/3 (4), epidermal growth factor, interferon, and plate
let-derived growth factor as mediators of the host response to
various disease states or as part of pathological processes.
The ability to lyse neoplastic and virus-transformed cells in
vitro and in vivo is a property of TNF (3) and is of great interest
in view of its possible use in cancer therapy. On the other hand,
because overproduction of this cytokine is associated with the
pathogenesis of many inflammatory diseases, approaches to
attenuating the action of TNF have been sought. The protective
Received 8/30/90; accepted 2/22/91.
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.
1This study is supported by NIH Grant Al-15614. J. E. B. is supported by a
Fellowship of the Ministry of Science and Technology of Brazil (CNPq).
2 To whom requests for reprints should be addressed, at the Department of
Medicine, New England Medical Center, 750 Washington St., Boston, MA
02111.
3The abbreviations used are: IL-1. interleukin 1; IL-6, interleukin 6; TNF-«,
tumor necrosis factor a; TGF-/3, transforming growth factor ß,;PKC, protein
kinase C; A-23187, calcium ionophore A-23187; PGE;. prostaglandin E2; ACT
D, actinomycin D; MnSOD, manganous Superoxide dismutase; PMA. phorbol
12-myristate 13-acetate; TNF LDSO,concentration of TNF/ACT D necessary to
achieve 50% cell cytotoxicity.
effect afforded by preadministration of IL-1 (5-7), TNF (810), epidermal growth factor, TGF-a, or TGF-/3 ( 11-12) usually
24 h before lethal doses of TNF, bacteria, or some inflammatory
agents has been demonstrated in various in vivo and in vitro
models.
The mechanisms which protect target cells from being killed
by TNF remain unclear. Previous reports (13-15) have shown
that activation of PKC (16) plays a role in the modulation of
TNF sensitivity, as suggested by the effect if its inducers PMA
and A-23187. However, it is uncertain whether cytokine-induced resistance is also dependent on PKC activation. Further
more, the kinases associated with the majority of cytokine
receptor-mediated responses remain to be defined. The present
experiments were undertaken to study how IL-1/3, IL-6, TNFa, and TGF-,0 induce an increase in cell resistance to TNF
cytotoxicity in murine L929 cells. Because TGF-/3 possessed
marked activity to protect these cells from the action of TNF,
additional experiments were carried out to evaluate the inter
actions between TGF-/3 and PKC inducers. Furthermore, in
view of the evidence from a previous study (17) that sensitivity
of cells to the lytic effects of TNF is increased by cell arrest in
mitosis, we also investigated the effect of cytokines and other
pharmacological agents on the L929 cell cycle to determine
whether cell resistance to TNF would be a cell cycle-linked
process. Our data suggest that increased cellular resistance
induced by these cytokines and PKC activators correlates with
the accumulation of cells in GI, whereas the increased sensitivity
induced by PGE2 and cholera toxin is associated with the
transition of cells from the G, to the G2 + M phase of the cell
cycle.
MATERIALS
AND METHODS
Reagents. Recombinant human IL-1/3 was the kind gift of Dr. Alan
R. Shaw (Glaxo Institute of Molecular Biology, Geneva, Switzerland).
Recombinant human TNF-a, TGF-/3, and IL-6 were provided by Genentech, Inc. (South San Francisco, CA). PMA (Sigma, St. Louis, MO)
and A-23187 (Calbiochem, La Jolla, CA) were dissolved in dimethyl
sulfoxide and further diluted in cell culture medium. The control
dilutions with dimethyl sulfoxide or alcohol had no effect in our
experiments. Actinomycin D was obtained from Calbiochem, and chol
era toxin was from List Biological (Campbell, CA). Prostaglandin E2
was purchased from Sigma, and a stock solution of 50 Mg/ml was
prepared by adding 1.0 ml of absolute ethanol followed by 19 ml of
tissue culture medium. A stock solution of 1.0 HIM indomethacin
(Sigma) was prepared in 0.1 M Tris buffer (pH 8.0) and was further
diluted with culture medium at the time of use.
Cell Culture. L929 mouse fibrosarcoma cells (CCL1) were purchased
from the American Type Culture Collection (Rockville, MD). Cells
were maintained in culture using RPMI 1640 supplemented with 5%
(v/v) heat-inactivated fetal calf serum (HyClone, Logan, UT) plus 2
mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 100 units/ml penicillin G, and 100 Mg/ml streptomycin
sulfate at 37°Cin a 5% CO2-humidified air incubator. Cells were washed
with saline, detached using 0.25% (v/v) trypsin and 20 Mg/ml EDTA,
and then resuspended in complete medium at a density of 50 x 10"
2379
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CYTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXICITY
cells/ml. \lillois (100 n\) were dispensed into 96-well flat-bottomed
microtiter plates, and the cells were then allowed to adhere for 3-4 h
in a 5% CO2 humidified air incubator. Thereafter, 10-^1 aliquots of
different concentrations of cytokines dissolved in complete medium
were added to the wells.
L929 Cell Cytotoxicity Assay. The assay was performed as described
by Flick and Gifford (18) with modifications. Briefly, 5 x IO4 L929
cells/well were incubated in 100 ^1 of RPMI containing 5% fetal calf
serum in 96-well plates overnight. Subsequently, the medium was
removed and replaced with 100 p\ RPMI containing 2.5% fetal calf
serum, supplemented with 5 /jg/ml actinomycin D (control) and serial
dilutions of TNF. Each dilution was performed in quadruplicate. The
plates were then incubated for a further 18-20 h. The plates were
agitated on a plate shaker the next day, and the medium was removed
by suction. The remaining cells were reincubated for an additional 3-4
h in RPMI containing 500 jig/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma). The medium was aspirated, and
100 fii of 2-propanol were added to each well to dissolve the formazan
crystals. The absorbance of each well was determined using a Bio-Rad
model 3550 microplate reader. Reproducibility in the quadruplicate
experiments was 5-15%. The percentage of cell survival was calculated
as
RESULTS
Dose-Response and Time Course of Cytokine-mediated In
creases in Resistance to TNF. Pretreatment of L929 cells with
the cytokines IL-1, IL-6, TNF, or TGF-0 induced a significant
(P < 0.05) increase in the dose of TNF/ACT D required to kill
50% of the cells (Fig. 1). The ability of these cytokines to reduce
the TNF cytotoxic effects was time and dose dependent. The
maximal effect of IL-1 was observed in cells incubated for 12 h
at a dose of 10 ng/ml (Fig. \A). The percentage of increase in
the TNF LD50 in cells pretreated with IL-6 was similar to that
of IL-1, but the effect was most pronounced after 24 h of
treatment (Fig. IB). The addition of TNF itself before chal
lenging cells with the combination TNF/ACT D also signifi200
150
too
Mean absorbance wells at a particular dose of TNF x 100
Mean absorbance wells in control
50
Dose-response curves were calculated from the percentage of cell sur
vival versus logio dilution using the least squares method. One unit of
TNF was defined as TNF LD50 (5 ¿ig/ml).The change in the cell
survival due to the treatment with cytokines or other reagents is
expressed as a percentage of increase in the TNF LD50 over the value
in control. In most experiments, the TNF LD50 for the cells in control
ranged from 0.3 to 0.6 ng/ml TNF. Comparison between experimental
groups was done by Student's t test, and significance was defined as P
200
150
too
50
< 0.05.
Flow Cytometric Analysis and Cell Cycle Distribution. The flow
cytometric assay was performed after staining of DNA utilizing the
propidium iodide method (19). Briefly, exponentially growing L929
cells were treated with the cytokines and other reagents for 6 h. The
cells were washed, and then trypsin-EDTA (Irvine Scientific, Santa
Ana, CA) was added for 1 min. The cells were suspended in medium
and centrifuged at 500 x g for 5 min, and the pellet was resuspended
in 2 ml phosphate-buffered saline (20 IHMsodium phosphate-150 mM
sodium chloride, pH 7.2). The cell suspension was fixed in 5 ml 95%
ethanol. The fixed cell suspension was allowed to stand for 30 min at
room temperature and was subsequently stored at 4°C.Before staining,
400
300
200
100
the sample was centrifuged, and I ml trypsin-EDTA was added. After
incubation for 2 min, the trypsin was inactivated by the addition of 2
ml medium. Cells were again centrifuged and stained by the addition
of I ml propidium iodide staining solution (50 Mg/ml propidium iodide,
100 Mg/ml RNase A, and 0.1% Triton X-100 in phosphate-buffered
saline). FACScan flow cytometric analysis was performed using a
Becton Dickinson immunocytometry system. The number of cells in
GI, S, and G2 + M compartments was obtained using the sum of
rectangles model. The experiments were repeated at least twice, and
10,000 cells were counted per sample.
|'ll| I Immillilo Incorporation Assay. Cells (5 x 104/well) were cul
tured overnight in 100 .//Iof medium in 96-well microtiter plates in a
humidified, 5% CO2 atmosphere at 37°C.The next day, cytokines or
other experimental reagents were added, and the cells were pulsed with
[3H]thymidine, 0.5 ^Ci/well (New England Nuclear, Boston, MA). Four
h later, cells were harvested onto paper filters with a cell harvester
(Cambridge Technology, Cambridge. MA). Incorporated radioactivity
was determined by liquid scintillation, and the results were expressed
as cpm ±SE of quadruplicate wells.
800
600
400
200
-6h
-12
h
-24
h
Time of treatment
Fig. 1. Dose response and time course of the increase in cell resistance to
TNF/ACT D cytotoxicity mediated by the cytokines IL-10 (A), IL-6 (A), TNF-o
(C), and TGF-fi (D). Exponentially growing cultures of L929 cells were pretreated
with a selected cytokine at the indicated concentrations and times followed by the
TNF killing assay. The percentage of increase in TNF LD50 was calculated by
comparing the values obtained from untreated control cells to those from cytokine-treated cells. Columns, means from three independent experiments; bars,
SE. Each value was significantly different (/' < 0.05) as compared with the
noncytokine-treated control response.
2380
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CYTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXICITY
cantly protected L929 cells. The percentage of increase in TNF
LD50 in cells pretreated with TNF alone for 6 h ranged from
83 to 279%. However, in cells incubated for 24 h, the increases
ranged from 79 to 193% (Fig. 1C), indicating a moderate
decrease in the acquired protection induced by TNF. TGF-ß
treatment induced a dramatic and rapid augmentation in de
fense against TNF cytotoxicity (Fig. 10). Cells incubated with
5 ng/ml for only 6 h increased the TNF LD50 by 620%. The
profiles of protection were similar in cells exposed to TGF-0
for 12 or 24 h.
Additional experiments showed that exposure of cells to
TGF-/3 plus TNF had an additive effect on the cell resistance,
but not IL-1 plus IL-6, IL-1 plus TGF-0, IL-1 plus TNF, IL-6
plus TNF, or IL-6 plus TGF-/3 (data not shown). On the other
hand, the incubation of a single cytokine simultaneously with
the combination TNF/ACT D did not increase the LD50 in the
assay, suggesting that the synthesis or suppression of interme
diate molecules is required to increase cell resistance. The
autocrine production of TNF, as has been described (20), was
not an explanation for the increase in resistance, since TNF
activity was not found in the conditioned medium of cells
culture in the presence of the cytokines used in this study.
TGF-ßActs Synergistically with Activators of PKC. As re
ported previously, activators of protein kinase C increase cell
survival through down-regulation of TNF surface receptors
(13-15). In view of these findings, we investigated the interac
tions of PKC activators on the TGF-0-mediated cellular resist
ance to TNF in cells exposed to either PMA or A-23187 (Fig.
2). The TNF LD50 in cells pretreated with PMA (Fig. 2A) for
7 h increased by only 193%, whereas in cells treated with the
combination of PMA plus TGF-/3 it increased to 1910%. Since
TGF-ßalone caused an increase of 932%, the data suggest a
synergy between TGF-ßand PMA. Similar synergistic effects
were also observed in cells treated with the combination of
TGF-ßplus A-23187 (Fig. 2B). In cells exposed to this combi
nation, the LD50 for TNF ranged from 3700 to 5500% (Fig.
2B).
A previous study demonstrated that exposure of cells to high
concentrations of PMA for 3 days depletes them of protein
kinase C activity (22). We examined the effect of treatment of
L929 cells with PMA (100 ng/ml) for 3 days on the subsequent
effect of TGF-/3-mediated protection (Fig. 3). As expected,
short-term addition of PMA did not increase the TNF LD50 in
cells pretreated for 3 days with PMA, as compared to control
cells. The protective effect of TGF-ßwas more pronounced in
cells treated with PMA for 3 days. However, the synergy
between TGF-ßand short-term PMA was significantly reduced,
as compared to control cells. Thus, the protective effects in
duced by TGF-ßplus PMA may be regulated through distinct
pathways or by isoenzymes of PKC resistant to depletion by
prolonged PMA treatment (16).
Cholera Toxin and PGE2 Block, whereas Indomethacin En
hances, TGF-0-mediated Increase in Cellular Resistance. Six h
of pretreatment of L929 cells with various concentrations of
PGE2 (Fig. 4A) resulted in a marked decrease in the ability of
the cells to respond to TGF-ß.In addition, cells pretreated with
PGE2 alone were more sensitive to TNF as compared to control
cells. This effect was significant (P < 0.05) at 100, 1000, and
2500 ng/ml (Fig. 4A). Prostaglandin modulation of TGF-ß
activity was also confirmed by treating cells with the cyclooxygenase inhibitor indomethacin (Fig. 4Ä).Concentrations rang
ing from 0.01 to 10 UM indomethacin caused an enhancement
of the ability of TGF-ßto increase cell resistance in a bimodal,
2000
O
1500 -
If
1000 500 -
7000 6000 5000 4000 K
3000 -
0.5
1.0
TGF-ß (ng/ml)
2.5
Fig. 2. TGF-(3-mediated increase in resistance to TNF cytotoxicity in cells
pretreated for 1 h with either 20 ng/ml PMA (A) or 0.2 »MA-23187 (B) and
then with TGI -.i at the indicated concentrations for an additional 6 h. A: •cells
treated with PMA for 7 h; D, untreated cell control with TGF-ßonly for 6 h; Q,
cells treated with PMA for 1 h and T(,I ,i for an additional 6 h. B: •.cells
treated with A-23187 for 7 h; D, untreated cell control with TGF-ßonly for 6 h;
D. cells treated with A-23187 for 1 h and TGF-ßfor an additional 6 h. The
percentage of increase in TNF 1.1),,, was calculated by comparing the values
obtained from untreated control cells to those from cytokine-treated cells. Col
umns, means from three independent experiments; bars, SE. Each value shown is
significant at P < 0.05.
1600 1200 800 -
«too-
Control
PMA treated
Fig. 3. TGF-ß-and PMA-mediated increase in cell resistance to TNF cytotox
icity in cells exposed to prolonged PMA treatment. L929 cells were first cultured
for 3 days in regular medium containing 100 ng/ml PMA (I'M.l treated) or
0.25% dimethyl sulfoxide vehicle (Control) and then seeded into 96-well microtiler plates. The next day, cells were pretreated for 1 h with 10 ng/ml PMA and
then with 1 ng/ml TGF-0 for an additional 6 h. •cells treated with PMA for 7
h; D, untreated cell control with TGF-/3 only for 6 h; Q, cells treated with PMA
for 1 h and TGF-/3 for an additional 6 h. The percentage of increase in TNF 1.1),„
was calculated by comparing the values obtained from untreated control cells to
those from cytokine/reagent-treated cells. Columns, means from three experi
ments; bars, SE. a, P < 0.005; ft, P < 0.05; c, P < 0.01 versus similar conditions
in control cells.
dose-dependent manner. However, 100 MMindomethacin re
duced the TGF-ßactivity. Indomethacin per re had no effect on
TNF cytotoxicity at these concentrations. Interestingly, when
L929 cells were treated with different doses of cholera toxin,
2381
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CYTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXICITY
800
PGE2 (Fig. 5, A and C) or cholera toxin (Fig. 5B). As shown
in Fig. 5, the increase in TNF LD50 induced by either TGF-0
or TGF-ßplus A-23187 was nearly abolished 6 h after the
treatment with PGE2 or cholera toxin. These data suggest that
the effects of A-23187 or TGF-/3 on protection against TNF
cytotoxicity can be down-modulated by cyclic AMP-stimulatory
factors.
Increased Proportion of Cells in G2 + M Phase of Cell Cycle
following PGE2 or Cholera Toxin Treatment. Fig. 6 shows the
cell cycle phase distributions of asynchronized growing cells
after 6 h of exposure to cytokines and/or other reagents. Dif
ferences no larger than 2% in the relative proportion of cells in
the G i and S phases were found in cells treated with IL-1, IL6, TNF, TGF-0, or TGF-0 plus the PKC activators, as com-
600
400
200
0
Ti
-200
o
n
PGEj + - +
TGF - + +
1
10
100
1000
2500
Pros tag Iand in E2 <ng/ml )
3000
I)
O)
e
e
w
o
n
0.01
0.1
1
10
Indome thac in <uM>
+•f
+-+
TGFPOE,1200900£00300-Tnn•-+_Bj.-+
100
+
-++ +rr-r-iI
+- +
800
M
600
400
200
-200
CT
TGF
-J
+ - +
TGF +
CT 2500
1
10
25
Cholera Toxin
50
100
<ng/ml>
Fig. 4. Changes in TGF-Ã-i-mediated increase in resistance to TNF cytotoxicity
in cells pretreated for 6 h with PGE2 (A), for l h with indomethacin (B), or for 6
h with cholera toxin (C) at the indicated concentrations. These treatments were
then followed by the addition of 1.0 ng/ml I (.1 .; for an additional 6 h. •(.•
D,
cells treated with PGE2 for 12 h; G, untreated cell control with TGF-/3 only for 6
h; •.cells treated with P(. I for 6 h and with !(,!.,
for an additional 6 h. B: •
cells treated with indomethacin (Indo) for 7 h; D, untreated cell control with
1111 ; only for 6 h; •.cells treated with indomethacin for 1 h and 1( ; I ; for an
additional 6 h. ( '.-•cells treated with cholera toxin (('/') for 12 h; D, untreated
1500
500
cell control with TGF-/3 only for 6 h; G, cells treated with cholera toxin for 6 and
TGF-tf for an additional 6 h. The percentage of change in TNF LD50 was
calculated by comparing the values obtained from untreated control cells to those
from cytokine/reagent-treated
cells. Positive scale, increases in LDM (increased
resistance); negative scale, decreases in 1.1><,>(increased sensitivity). Data of one
representative experiment from four independent assays.
which shares with PGE2 the ability to induce cyclic AMP, there
was also inhibition of TGF-|3-mediated reduction of TNF cy
totoxicity (Fig. 4C). We also observed that the cells exposed to
cholera toxin are more sensitive to TNF/ACT D than are
control cells. Cholera toxin and PGE2 themselves were not
toxic to L929 at the concentrations used in this study.
Additional experiments were undertaken to determine the
sensitivity of cells pretreated for 6 h with TGF-ßor TGF-/3 plus
A-23187 and subsequently incubated for an additional 6 h with
n
TCF/fl-23187
PGE2
0.5
TGF-p
1.0
<ng/ml>
2.5
Fig. 5. Changes in resistance to TNF cytotoxicity in cells pretreated for 6 h
with either TGF-Ö or TGF-/3 plus 0.1 JIM A-23187 at the indicated concentrations
and then with 1.0 Mg/ml PGE2 (A, C) or 100 ng/ml cholera toxin (B) for an
additional 6 h. A: Q, cells treated with TGF-,8 for 21 h; Q. untreated cell control
with PGE2 only for 6 h; •,cells treated with TGF-ß for 6 h and PGE¡ for an
additional 6 h. B: G, cells treated with TGF-0 for 12 h;H, untreated cell control
with cholera toxin (C7") for 6-h; H, cells with TGF-fi for 6 h and cholera toxin
for an additional 6 h. C: G, cells treated with TGF-ii and A-23187 for 12 h; G,
untreated cell control with PGE2 only for 6 h; O. cells treated with TGF-0/A23187 for 6 h and PGE2 for an additional 6 h. The percentage of change in TNF
LDso was calculated by comparing the values obtained from untreated control
cells to those from cytokine/reagent-treated
cells. Positive columns, increases in
LD50 (increased resistance); negative columns, decreases in LD50 (increased sen
sitivity). Columns, means from three independent experiments; bars. SE.
2382
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CYTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXICITY
800
600
400
ft
§
-200
THF
Gì
62
8
30
G2+M
8
CT
GÌ
41
8
33
G2+M 26
CHX
E
3
Z
PGE2
Gì
43
8
34
G2 + M 23
flCT D
HIT
C
Fig. 8. Changes in the cell resistance to TNF cytotoxicity in cells pretreated
for 6 h with 10 ng/ml cycloheximide (CHX), 1 nM puromycin (PURO), 1 Mg/ml
actinomycin D (ACT D), or 5 ^g/ml mitomycin (MIT C). The percentage of
change in TNF 1.))..,, was calculated by comparing the values obtained from
untreated control cells to those from drug-treated cells. Positive columns, increases
in LDM (increased resistance); negative columns, decreases in LD50 (increased
sensitivity). Each value is significantly different as compared with untreated
controls (P< 0.05).
I«
O>
JO
TGF + PMA
GÌ 57
PURO
pared to the control experiment cultured in medium containing
5% fetal calf serum. In cells treated with PGE2 or cholera toxin,
there was a higher proportion of cells in G2 + M and a
pronounced decrease in the proportion of cells in Gt. These
findings suggest that agents and cytokines leading to an increase
in cell resistance are likely to be associated with the arrest of
cells in d; on the contrary, agents leading to an increase in cell
sensitivity are associated with a marked enhancement of cells
in the G2 + M phase of the cell cycle. To confirm this hypoth
esis, cells were stimulated with the cytokines or other com
pounds in the presence of ['11|th\ midine during the initial 4 h
0>
OÃ-
Relative PI Fluorescence
Fig. 6. Cell cycle phase distribution (after 6 h of treatment) and [3H)thymidine
incorporation (within the initial 4 h of treatment) in asynchronous growing
cultures of L929 cells incubated with medium alone (.-I) or supplemented with 10
ng/ml IL-1 (B), 10 ng/ml IL-6 (C), 5 ng/ml TGF-,3 (D), 10 ng/ml TNF (£),5
ng/ml TGF-0 plus 10 ng/ml PMA (F). 5 ng/ml TGF-0 plus 0.2 «MA-23187 (C),
100 ng/ml cholera toxin (//) or 1.0 fig/ml PGE2 (/). DNA histograms were
generated on a FACScan using propidium iodide (19). The percentage of cells in
the GI, S, or GI + M compartments was obtained using the sum of rectangles
model. Columns, cpm of quadruplicate determinations; bars, SE. *, P < 0.05
venus control. CT, cholera toxin; PI, propidium iodide; TGF, TGF-/3; A-23, A23187.
of incubation (Fig. 6, columns). The treatment of cells with the
cytokines IL-1, IL-6, TNF, or TGF-0 (Fig. 6B-E) caused a
significant decrease in the incorporation of thymidine. The
combination of TGF-/3 plus PMA (Fig. 6F) did not change
thymidine incorporation, whereas the combination TGF-,0 plus
A-23187, cholera toxin, or PGE2 significantly increased the
uptake. Interestingly, the rates of thymidine incorporation
within 4-8 h of incubation were significantly (P< 0.05) reduced
in cells incubated in the presence of cytokines and/or other
reagents (Fig. 7), as compared with the control experiment
incubated with medium.
Inhibitors of Cell Cycle Progression Increase Cellular Resist
ance to TNF. To further study the relationship between cell
cycle progression and protection, we carried out experiments
to ascertain whether prior exposure of cells to cycloheximide,
puromycin, or actinomycin would inhibit cell growth predomi
nantly during the GI and S phases of the cell cycle (23, 24) and
thus interfere with the resistance of cells to TNF. As depicted
in Fig. 8, pretreatment of cells with these metabolic inhibitors
resulted in a significant increment in the TNF LD50, whereas
treatment with mitomycin, a DNA-intercalating agent that
causes cells to arrest in mitosis, reduced resistance.
DISCUSSION
Fig. 7. [3H]Thymidine incorporation during the 4-8-h time period in cultures
of L929 cells incubated with culture medium alone (control) or supplemented
with cytokines and/or other agents as described in Fig. 6. Columns, cpm of
quadruplicate determinations: bars, SE. All treatments resulted in significant
inhibition of thymidine uptake as compared to control (P < 0.05). CT, cholera
toxin.
Most cells are inherently resistant to the cytotoxic effects of
TNF (3, 11, 12). The molecular mechanism leading from a
resistant to a sensitive state remains unknown. The mechanism
by which a short exposure to some factors induces a transient
increase in the relative resistance of susceptible target cells to
the cytotoxicity of TNF is not fully understood. The results of
the current study provide evidence for a possible mechanism by
which prior administration of the cytokines IL-1, IL-6, TNF,
2383
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CYTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXICITY
or TGF-ßor various pharmacological agents for 12-24 h before
challenging murine L929 cells with a lethal dose of TNF/ACT
D can attenuate cellular death.
Several mechanisms of cellular regulation of the action of
TNF have been described. The activation of PKC leads to the
desensitization to TNF (13-15). We observed that treatment of
L929 cells with PMA or A-23187, as well as with the PKC
inhibitor sphingosine (21), enhances cell survival (25). Addi
tional experiments showed that either PMA or A-23187 to
gether with TGF-/3 acts in a synergistic manner to augment the
cell resistance. Despite these observations, it was shown that
TGF-/J continued to exert its protective effect on cells made
deficient in protein kinase C by prolonged incubation with
PMA, suggesting that molecular events associated with either
PKC activation or a non-PKC pathway may also protect cells
from death.
The treatment of cells with either TNF (26) or IL-1 (26, 27)
induces MnSOD, a mitochondrial enzyme involved in the scav
enging of Superoxide anions. Such activated oxygen species
have been proposed to be part of the mechanism of TNF
cytotoxicity since the overexpression of the MnSOD gene pro
tects cells from lethal injury by TNF (28). Hence, the protective
effects evoked by the TNF and IL-1 treatment on L929 cells
might be explained by the synthesis and scavenger activity of
MnSOD. On the other hand, the protective effects evoked by
IL-6 and TGF-/Õare likely to be independent of antioxidant
defenses because these cytokines are not inducers of MnSOD
(26).
Previous studies have shown that following exposure of target
cells to TNF, most of them die at late stages of mitosis or soon
after cytokinesis (17, 29). Consistent with these observations,
it was also noted that drugs which cause cells to arrest in
mitosis, such as vinblastine, Colcemid (17), or mitomycin (29),
potentiate sensitivity to TNF. We have shown in the present
study that both PGE2 and cholera toxin are also capable of
augmenting sensitivity to TNF. Most importantly, it was ob
served that soon after the addition of these compounds, an
increased accumulation of cells in G2 + M occurs. DNA syn
thesis is stimulated during the initial 4 h of the incubation and
thereafter is inhibited. It has been established in other studies
(30) that both PGE2 and cholera toxin promote cells to prolif
erate through the activation of adenylate cyclase. A sustained
cAMP elevation, on the other hand, inhibits the proliferation
and induces the differentiation of many mammalian cells (30).
It is also interesting that TNF increases the synthesis of prostaglandins in a wide variety of cells (3). Our findings confirm
and further extend the conclusion of earlier studies (17, 29)
that TNF cytotoxicity is associated with DNA replication and
cell division and that in the M-phase cells are most sensitive to
TNF action.
Since the arrest of cells in G2 + M makes them more
vulnerable to TNF, the arrest of cells in G, of the cell cycle in
response to IL-1, IL-6, TNF, or TGF-/J makes them more
resistant. In fact, analysis of viability and of the relative pro
portions of cells at various cell cycle phases in cultures of Lcells (17) or L929 cells4 treated with TNF has provided evidence
early and decisive biochemical events in G, that prepare them
to enter into the cycle (23, 24). Indeed, several studies have
shown that IL-1 (1, 31), IL-6 (2, 32), TNF (3, 11), and TGF-/ÃŽ
(4, 33) exert a strong inhibition on growth of a variety of cell
lines. Furthermore, the reduction of c-myc transcription by
TGF-/3 (33) and TNF (34), the suppression of ornithine decarboxylase activity by IL-1 and TNF (31), the induction of inter
ferons and the enzyme 2',5'-oligoadenylate synthetase by IL-1,
IL-6, and TNF (32, 35), and the down-regulation of mitogen
receptors by TGF-ß(36) have been described as mechanisms by
which these cytokines mediate growth inhibition on a variety
of tumor cell lines and normal tissue cells.
More direct evidence that protection is associated with a GI
phase growth arrest comes from results in this study which
show that prior exposure of cells to cycloheximide, puromycin,
or actinomycin D also prevents cell death by TNF/ACT D.
These results further support our hypothesis that a cell popu
lation resistant to TNF is generated in response to either the
cytokines or other agents capable of blocking the early events
in G, phase (23, 24). According to this concept, exponentially
growing cells, which are not affected by a cytokine-induced
protection, enter into S phase as soon as they are stimulated by
the growth factors present in fetal calf serum, which is added
simultaneously with the combination TNF/ACT D. Thereafter,
ACT D renders these cells more sensitive to TNF cytolytic
effects by blocking the synthesis of TNF-induced repair proteins
(3, 18) and by arresting and synchronizing cells in the G2 + M
compartment.
In conclusion, our results suggest that cytokines IL-1, IL-6,
TNF, and TGF-/3 increase cell resistance in TNF-susceptible
cells by inducing growth arrest in the GI phase of the cell cycle,
as confirmed by a significant reduction in cell DNA synthesis
after cytokine treatment. In contrast, the enhanced cellular
sensitivity which occurs when cells are exposed to either PGE2
or cholera toxin is caused by an augmentation of DNA synthesis
and passage of cells from the GI to the G2 + M phase where
they are maximally susceptible to cytotoxic effects of TNF.
ACKNOWLEDGMENTS
We thank Dr. Michael A. Palladino, Jr., for his support. We also
thank Scott F. Orencole and Jean Hermann for their excellent technical
assistance and Silvia Judd for help in preparing the manuscript.
REFERENCES
that cells in G2 are more resistant to TNF. Accordingly, we
observed that exposure of cells to either cholera toxin or PGEi
alters the protective effect of TGF-ßpresumably because these
compounds promote a transition of cells from Gìto G2 + M.
Together these observations suggest that the effective action of
these cytokines is dependent upon their ability to block the
4 Unpublished observations.
1. Dinarello. C. A. lnterleukin-1 and its biologically related cytokines. Adv.
Immunol., 44: 153-205, 1989.
2. Heinrich, P. C., Castelli, J. V., and Andus, T. Interleukin-6 and the acute
phase response. Biochem. J., 265:621-636. 1990.
3. Rosenblum, M. G., and Donato, N. J. Tumor necrosis factor o: a multifaced
peptide hormone. Crit. Rev. Immunol., 9: 21-44, 1989.
4. Keski-Oja, J., Leof, E. B., Lyons, R. M., Coffey, R. J., and Moses, H. L.
Transforming growth factors and control of neoplastic cell growth. J. Cell.
Biochem., 33: 95-107, 1987.
5. Holtmann, H., and Wallach, D. Down regulation of the receptors for tumor
necrosis factor by inlerleukin 1 and 4(¡-phorbol-12-myristate-13-acetate. J.
Immunol., 139: 1161-1167, 1987.
6. Holtmann, H.. Hahn, T., and Wallach, D. Interrelated effects of tumor
necrosis factor and interleukin-1 on cell viability. Immunobiology, 777: 722, 1988.
7. van Der Meer, J. W. M.. Barza, M., Wolff, S. M., and Dinarello, C. A. A
low dose of recombinant interleukin-1 protects granulocytopenic mice from
lethal Gram-negative infection. Proc. Nati. Acad. Sci. USA, 85: 1620-1623,
1988.
8. Wallach. D. Preparations of lymphotoxin induce resistance to their own
cytotoxic effect. J. Immunol.. 132: 2464-2469, 1984.
9. Wallach, D., Holtmann. H., Engelmann, H., and Nophar, Y. Sensitization
and desensitization to lethal effects of tumor necrosis factor and interleukin1. J. Immunol., 140: 2994-2999, 1988.
2384
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
CVTOKINE-INDUCED
RESISTANCE TO TNF CYTOTOXIC'ITY
10. Lehmann, V., Freudenberg, M. A., and CaÃ-anos, C. Lethal toxicity of
lipopolysaccharide and tumor necrosis factor in normal and D-galactosaminetreated mice. J. Exp. Med., 165: 657-663, 1987.
11. Surgarman. B. J., Lewis, G. D., Eessalu, T. E.. Aggarwal. B. B., and Shepard.
H. M. Effects of growth factors on the antiproliferative activity of tumor
necrosis factor. Cancer Res., 47: 780-786, 1987.
12. Shepard, H. M., and Lewis, G. D. Resistance of tumor cells to tumor necrosis
factor. J. Clin. Immunol., 8: 333-341, 1988.
13. Aggarwal. B. B., and Eessalu, T. E. Effect of phorbol esters on downregulation and redistribution of cell surface receptors for tumor necrosis
factor-«.J. Biol. Chem.. 262.- 16450-16455. 1987.
14. Johnson. S. E.. and Baglioni. C. Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C. J. Biol.
Chem.. 263: 5685-5692, 1988.
15. Sheurich, P.. Kobrich, G., and Pfizenmaier, K. Antagonistic control of tumor
necrosis factor receptor by kinases A and C. J. Exp. Med., 170: 947-958,
1989.
16. Nishizuka, Y. The molecular heterogeneity of protein kinase C and its
implications for cellular regulation. Nature (Lond.), 334: 661-665, 1988.
17. Darzynkiewicz, Z., Williamson. B., Carswell, E. A., and Old. L. Cell cyclespecific effects of tumor necrosis factor. Cancer Res.. 44: 83-90, 1984.
18. Flick, D. A., and Gifford, G. E. Comparison of in vitro cell cytotoxic assay
for tumor necrosis factor. J. Immunol. Methods, 68: 167-175, 1984.
19. Clevenger, C. V., Bauer, K. D., and Epstein, A. L. A method for simultaneous
nuclear immunofluorescence and DNA content quantitation using monoclo
nal antibodies and flow cytometry. Cytometry, 6: 208-213, 1985.
20. Rubin, B. Y.. Anderson. S. L., Sullivan. S. A.. Williamson. B. D.. Carswell,
E. A., and Old, L. J. Nonhematopoietic cells selected for resistance to tumor
necrosis factor produce tumor necrosis factor. J. Exp. Med., 164: 13501355, 1986.
21. Merril, A. H., and Stevens, V. L. Modulation of protein kinase C and diverse
cell functions by sphingosine—a pharmacologically interesting compound
linking sphingolipidsand signal transduction. Biochim. Biophys. Acta, 1010:
131-139, 1989.
22. Rozengurt, E., Rodriguez-Pena, A.. Coombs. M.. and Sinnet-Smith, J. Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells:
role of CaJ*-sensitive phospholipid-dependent protein kinase. Proc. Nati.
Acad. Sci. USA. 81: 5748-5752, 1984.
23. Pardee, A. B. G, events and regulation of cell proliferation. Science (Wash
ington DC). 246:603-608. 1989.
24. Rossow, P. W., Riddle, V. G., and Pardee, A. B. Synthesis of labile, serum-
dependent protein in early G, controls animal cell growth. Proc. Nati. Acad.
Sci. USA, 76:4446-4450, 1979.
25. Belizario, J. E., and Dinarello, C. A. Role of PKC on cytokine-induced
resistance to TNF cytolytic effects. Cytokines, /: 81, 1989.
26. Wong. G. H. W.. and Goeddel. D. V. Induction of manganous Superoxide
dismutase by tumor necrosis factor: possible protective mechanism. Science
(Washington DC). 242:941-944. 1988.
27. Masuda, A., Longo, D. L., Kobayashi, Y., Appela, E., Oppenheim, J. J., and
Matsushima, K. Induction of mitochondria! manganese Superoxide dismutase
by ¡nterleukin-1.FASEB J., 2: 3087-3091, 1988.
28. Wong, G. H. W., Elwell, J. H., Oberley. L. W., and Goeddel, D. V.
Manganous Superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell, 58: 923-931, 1989.
29. Coffman, F. C., Green, L. M., and Ware, C. F. The relationship of receptor
occupancy to the kinetics of cell death mediated by tumor necrosis factor.
Lymphokine Res., 7: 371-383, 1988.
30. Lohmann, S. M., and Walter, U. Regulation of the cellular and subcellular
concentrations and distribution of cyclic nucleotide-dependent protein ki
nases. Adv. Cyclic Nucleotide Protein Phosphorylation Res., 18: 63-76,
1984.
31. Endo, Y.. Matsushima. K., Onozaki, K., and Oppenheim, J. J. Role of
umiliimi' decarboxylase in the regulation of cell growth by 111 and tumor
necrosis factor. J. Immunol., 141: 2342-2348, 1988.
32. Kohase. M., Henriksen-DeStefano, D., May, L. T., Vilvek, J., and Sehgal,
P. B. Induction of rfrimerferon by tumor necrosis factor: a homeostatic
mechanism in the control of cell proliferation. Cell, 45: 659-666, 1986.
33. Pietenpol, J. A., Stein, R. W., Moran. E.. Yaciuk. P.. Schlegel. R.. Lyons,
R. M., Pittelkow, M. R., Munger, K., Howley, P. M., and Moses, H. L.
TGF-rf inhibition of c-myc transcription and growth in keratinocytes is
abrogated by viral transforming proteins with pRB binding domains. Cell,
61: 777-785, 1990.
34. Yarden, A., and Kimchi, A. Tumor necrosis factor reduces c-myc expression
and cooperates with interferone in Hela cells. Science (Washington DC),
234: 1419-1421, 1986.
35. Dcfilippi. P.. Poupart, P.. Tavernier. J., Fiers, W., and Content, J. Induction
and regulation of mRNA encoding 26-kDa protein in human cell lines treated
with recombinant human tumor necrosis factor. Proc. Nati. Acad. Sci. USA,
«4:4557-4561. 1987.
36. Battegay. E. J.. Raines. E. W., Seifen, R. A.. Bowen-Pope. D. F., and Ross.
R. TGF-rf induce bimodal proliferation of connective tissue cells via complex
control of an autocrine PDGF loop. Cell, 63: 515-524. 1990.
2385
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.
Interleukin 1, Interleukin 6, Tumor Necrosis Factor, and
Transforming Growth Factor β Increase Cell Resistance to
Tumor Necrosis Factor Cytotoxicity by Growth Arrest in the G 1
Phase of the Cell Cycle
José E. Belizario and Charles A. Dinarello
Cancer Res 1991;51:2379-2385.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/51/9/2379
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.