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Regular Articles |
§
From the Cancer Immunotherapy Research
Laboratory,*
Ecole Pratique des Hautes Etudes, and
INSERM U517, Faculty of Medicine, Dijon, France; CNRS,
Villejuif, France; Institute of Biology,
INSERM U419,
Nantes, France; and the G.F. Leclerc Anticancer Research
Center,§
Dijon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is a cytotoxic molecule originally described as a mediator of macrophage cytotoxicity.13,14 Many cell types, including tumor cells,15-17 synthesize NO, the production of which in large quantities involves inducible NO synthase (iNOS; NOS type II). The NO induced has both immunosuppressive15 and antitumor14,17,18 properties. Therefore, NO synthesis must be under tight control. The iNOS expression appears to be regulated at several levels: induction,19 transcriptional and posttranscriptional levels,11,12 and posttranslational level.12 TGF-ß has emerged as one of the most potent NO down-regulatory cytokines described so far. Thus, autocrine or paracrine production of TGF-ß may serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-ß production may lead to more effective therapeutic strategies in treating cancer.
Using a syngeneic model of peritoneal carcinomatosis induced in BDIX rats with PROb colon tumor cells, we previously showed the involvement of TGF-ß1 in tumor-induced immunosuppression in vivo and the ability of an immunotherapeutic treatment to reverse TGF-ß1-mediated immunosuppression.3 In this model, an intraperitoneal treatment with a lipid A cures 30% of the rats. Tumor regression correlates with increased NO concentration in the lipid A-treated tumor nodules. In these nodules, tumor cells were identified as the NO-producing cells.20 In the present study, we investigated the role of TGF-ß1 in the regulation of NO synthesis in tumor nodules during tumor development and lipid A-mediated tumor regression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tumor cell clone DHD/K12-PROb (named PROb) was isolated from a colon carcinoma induced by 1,2-dimethylhydrazine in a BDIX rat.21 These cells as well as the PROb cells transfected with an expression vector coding (PROb Bulk) or not (PROb Neo) for TGF-ß1 antisense mRNA and the two PROb cell clones (clone 3' and clone 13) were cultivated in Ham F10 medium (BioWhittaker, Fontenay-sous-Bois, France) supplemented with 10% fetal bovine serum (FBS) from Gibco BRL (Cergy Pontoise, France), and 40 µg/ml gentamycin. The latter is referred to as complete medium. EC 219 cells, a rat endothelial established cell line22 kindly given by Dr. L. Juillerat (Institute of Pathology, Lausanne, Switzerland), and mink lung epithelial cells (CCL 64, American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker) supplemented with 10% FBS, 2 mmol/L L-glutamine (BioWhittaker) plus 40 µg/ml gentamycin. To adhere, EC 219 cells were cultured on collagen-coated flasks.22 The cells were checked monthly and before each in vivo experiment to ensure they were free of mycoplasma contamination.
Peritoneal Carcinomatosis
Peritoneal tumors were induced in inbred BDIX rats (3- to 5-month-old males or females) purchased from IFFA Credo (l'Arbresles, France) by injection i.p. of 1 x 106 syngeneic PROb cells. Fourteen days later all control rats exhibited solid nodules of several millimeters and hemorrhagic ascites; they died of their tumors between weeks 8 and 12.3,15,20
Immunotherapy
The immunostimulant OM 174 (OM Laboratories, Meyrin, Switzerland) is the sodium salt of a purified lipid A analog from Escherichia coli. It consists of a hydrophilic diphosphorylated ß (1, 6) glucosamine disaccharide in which N-2 and N-2' carry a hydrophobic group, respectively (R)-3-hydroxytetradecanoyl and (R)-3-dodecanoyloxytetradecanoyl.
The treatment started 14 days after the tumor cell injection (day 0), when all the rats exhibited tumor nodules 2–5 mm in diameter. It consisted of five i.p. injections at the dose of 1 mg/kg, administered biweekly in the treated rats. The control rats received the same number and frequency of i.p. injections of the vehicle used to inject OM 174 (NaCl 9 g/l). At 15, 22, 29, 35, and 42 days after the tumor cell injection, three rats from each group were killed and autopsied. Peritoneal nodules were collected and either put immediately into liquid nitrogen for 15 minutes and kept at -80°C for iNOS Western blotting and TGF-ß1 dosages, or put in 10% buffered formalin for immunohistochemistry study.
Preparation of Tumor and PROb Cell Extracts
Tumor nodules collected at different times during the treatment were homogenized at 4°C with an ultra-turrax homogenizer in cold buffer A (50 mmol/L Tris-HCl, pH 7.4, 1 mmol/L EDTA, 10 mg/l antipain, 10 mg/l leupeptin, 10 mg/l soybean trypsin inhibitor, 10 mg/l pepstatin A, 10 mg/l chymostatin, 100 mg/l phenylmethylsulfonyl fluoride) using 4 ml of buffer per gram of tumor. The homogenate was centrifuged at 105,000 x g for 1 hour at 4°C and the supernatant (cytosol fraction) was collected and frozen at -20°C.
PROb cell extracts were prepared as follows: 4 x 106 cells were plated in 75-cm2 flasks and cultured for 4 days in complete medium. Then the cells were washed 3 times with serum-free medium and left at 37°C for 24 hours. The medium was removed and the cells were detached with a mixture of trypsin (2.5 mg/ml) plus EDTA (10 mg/ml) and centrifuged 10 minutes at 100 x g. The lysis of the pellet was obtained by adding 500 µl of lysis buffer (0.2% triton X-100, 10% pepstatin A, 10% aprotinin, 10% antipain dihydrochloride in PBS). The pellet was homogenized and left in the lysis buffer at 4°C for 45 minutes, then centrifuged 10 minutes at 27,500 x g. The PROb cell extracts (cytosol fraction) were collected and frozen at -20°C. Before TGF-ß1 determination, the protein content of tumor samples and PROb cell extracts were determined using the DC protein assay kit from Bio-Rad (Ivry sur Seine, France).
TGF-ß1 Assay
Active TGF-ß1 was measured using the enzyme-linked immunosorbent assay (ELISA) Quantikine human TGF-ß1 kit (R & D Systems, Abingdon, UK). In some experiments, the samples were acidified by the addition of HCl 1 N to give a pH of 2.0 to activate latent TGF-ß1, left 10 minutes at room temperature, and neutralized with 1.2 N NaOH/0.5 M HEPES. The kit does not detect TGF-ß2 or TGF-ß3 but allows the quantification of rat TGF-ß1, as indicated in the manufacturer's protocol.
Western Blotting
For iNOS detection, tumor extracts were separated in 7.5% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked by overnight incubation at 4°C with 10% nonfat milk in PBS containing 0.1% Tween 20. Then they were incubated with a 1/500 dilution of rabbit anti-inducible or anti-constitutive NOS antiserum.23 The membranes were washed and then incubated with anti-rabbit IgG conjugated with horseradish peroxidase and bands were revealed using an enhanced chemiluminescence (ECL) kit (Amersham, Les Ulis, France) and exposure to Hyperfilm at ambient temperature.
For TGF-ß receptor detection, PROb cell extracts and electrophoresis were performed as previously described.24 Marker proteins were Rainbow markers (Amersham). Western blotting of 10% polyacrylamide gels was done on Immobilon 0.45-µm membranes (Millipore, Saint Quentin, France) using a mini-protean II apparatus (Bio-Rad) at 100 volts for 1 hour at 4°C. The blots were processed as recommended by the antibody supplier and revealed with an ECL kit (Amersham) and Kodak films. Anti-TGF-ß RI was generously supplied by Drs. Miyazono and Heldin from the Ludwig Institute (Uppsala, Sweden)25 and the anti-TGF-ß RII was from Santa Cruz Biotechnology (Wembley, UK). Primary antibody dilutions were 1:2500 for anti-TGF-ß RI and 1:2000 for anti-TGF-ß RII. Secondary antibody dilutions were respectively 1:10,000 and 1:5000. The CCL64 mink epithelial cell line was used as positive control.
TGF-ß1 Receptor Affinity Labeling
For these experiments, cells were plated at 1 x 106 per 10 cm diameter dish in 8 ml of DMEM plus 5% FBS. The next day, cells were rinsed with PBS and incubated in serum-free DMEM for 3 hours at 37°C. Cells were affinity-labeled using 75 pmol/L 125I-TGF-ß1 (NEN, Les Ulis, France) in presence or absence of 4 nmol/L unlabeled human recombinant TGF-ß1 (R & D Systems) in PBS with 1 mg/ml BSA for 1 hour at 4°C. TGF-ß1 binding sites were cross-linked to 125I-TGF-ß1 using 0.2 mmol/L disuccinimidyl suberate (Pierce, Rockford, IL) in PBS for 20 minutes. Cells were collected by scraping, centrifuged in PBS, and solubilized in extraction buffer (10 mmol/L Tris-HCl, pH 7.2, 1 mmol/L EDTA, 1% Triton X-100, 1 mmol PMSF). Extracts were cleared by centrifugation and separated on 8% polyacrylamide gels under reducing conditions. For autoradiography, the dried gels were exposed to Kodak films.
Stimulation of the PROb and EC 219 Cells
The PROb cell suspension was adjusted to 0.5 x
106
cells/ml of complete medium and 100 µl/well were
seeded in quadruplicate, in 96-well plates for 24 hours at 37°C. The
cells were then washed twice with Ham F10 medium and 200 µl of fresh
Ham F10 medium, either containing or not containing the cytokines, were
added. The EC 219 cell suspension was adjusted to 0.15 x
106
cells/ml of complete medium and 1 ml/well were seeded
in 24-well plates coated with collagen for 24 hours at 37°C. Then,
the cells were washed twice with DMEM medium and 1 ml of fresh complete
medium, either containing or not containing the cytokines, was added.
The cytokines used were recombinant human IL-1ß (1 ng/ml) kindly
given by Dr. A. Minty from Sanofi (Labège, France), recombinant
human TNF-
(50 ng/ml) given by Dainippon (Tokyo), rat IFN-
(100
U/ml) generously given by Roussel Uclaf (Romainville, France) and
recombinant human TGF-ß1 (0.5 to 10 ng/ml) obtained from Genzyme
(Paris). In some experiments, an anti-human TGF-ß type II receptor
neutralizing antibody (R & D Systems) or NO donors: glyceryl trinitrate
(GTN, Merck, Nogent sur Marne, France) and the diethylamine NO complex
(DEA/NO, Coger, Paris), were added. The plates were incubated at 37°C
for either 48 hours (PROb cells) or 24 hours (EC 219 cells). Cell
supernatants were collected and frozen at -20°C for subsequent NO
dosage.
Quantitation of NO Production
NO was measured as the sum of the nitrite (NO2-) and nitrate (NO3-) content. Nitrate was reduced to nitrite using nitrate reductase (Boehringer Mannheim, Meylan, France) in the presence of NADPH. The samples were then assayed for NO2- content by the Griess assay26 as described elsewhere.15 In some experiments, 5 mmol/L of NG-monomethyl-L-arginine acetate (NMMA) purchased from Calbiochem (San Diego, CA) was added.
Immunohistological Studies
As previously described,7 tumor sections (3 µm) were deparaffinized and endogenous peroxidase activities were quenched in 0.3% (v/v) hydrogen peroxide in absolute methanol for 30 minutes. Sections were incubated with an anti-TGF-ß1 antibody (R & D Systems) at 4°C overnight. After washing in PBS, a peroxidase-conjugated antibody was added for 1 hour at 37°C. The peroxidase reaction was developed with the AEC kit (Vector Laboratories, Burlingame, CA). No immunostaining was detected when the slides were processed either in the absence of the primary antibody or in the presence of nonimmune serum. After hematoxylin counterstaining, the slides were permanently mounted in an aqueous medium (Aquatex) and analyzed for the presence and the distribution of the immunostaining. For the morphological study, sections were stained with hematoxylin-eosin-saffron (HES).
PROb Cell Transfection
The phCMV vector was kindly provided by Dr. R. Breathnach (INSERM U463, Nantes, France) and the pGEM-3Z vector was obtained from Promega (Charbonnières, France). The rat cDNA was transferred from BS KS pRTGF-ß1 plasmid (No. 63197, TCC) to the phCMV plasmid in a two-step process. First, the cDNA was excised from pRTGF-ß1 using HindIII and XbaI and ligated in the multicloning site of the pGEM-3Z plasmid. Then, the cDNA was excised from this plasmid using KpnI and HindIII and ligated, in an antisense orientation, in the multicloning site of the phCMV plasmid under control of the cytomegalovirus promoter. The phCMV plasmid contains the gene coding for geneticin resistance. Lipofectamine was used as recommended by the manufacturer (Gibco BRL) to transfer the phCMV TGF-ß1-antisense plasmid in the PROb cell line. Selection of the transfected cells was performed in complete medium containing 200 µg/ml neomycin (Gibco BRL). Surviving clones were isolated using cloning rings.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that NO concentration
(NO2- and NO3-)
increased up to fivefold above the basal level in PROb tumor nodules
from lipid A-treated rats.20
In the present study, we
analyzed the expression of iNOS protein by Western blotting in tumor
samples collected at different times during tumor growth and tumor
regression induced by the lipid A treatment. Using an anti-iNOS
antibody, virtually no immunoreactive band was observed at 125 kd in
tumor nodules from untreated rats. Conversely, in tumor nodules from
lipid A-treated rats, such an immunoreactive band was clearly visible
in all the samples; its intensity increased from day 15 to day 29 of
the treatment, then decreased after the last lipid A injection (Figure 1)
. Western blot analysis of the same gel
with anticonstitutive NOS antibody did not show any immunoreactive
bands, whereas immunoreactive bands of comparable intensity were
obtained for all the samples, with an anti-ß actin antibody used as
loading control (not shown).
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Because TGF-ß1 was demonstrated to inhibit NO synthesis in a
variety of normal cells, we investigated its expression in PROb tumor
nodules. Total TGF-ß1 concentration was measured by ELISA in
peritoneal tumor nodules collected at different times during tumor
growth and tumor regression induced by the lipid A treatment. After one
week of lipid A treatment, TGF-ß1 concentration was significantly
lower in treated compared to untreated tumor nodules. Decreased
TGF-ß1 intratumoral level persisted 2 weeks after the last lipid A
injection (Figure 2)
. Comparable results
were observed for active TGF-ß1, but concentrations were lower.
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Histological and immunohistological studies of treated and
untreated tumor nodules collected 28 days after the tumor cell
injection showed that untreated tumor nodules were made up mainly of
epithelial neoplastic cells, associated with a few inflammatory cells,
fibroblasts and macrophages. Some necrotic areas occurred in the center
of the nodules (Figure 3A
1).
In such nodules, 90–100% of the PROb tumor cells were strongly
stained by the anti-TGF-ß1 antibody, whereas macrophages,
lymphocytes, and fibroblasts remained unstained (Figure 3A
2). In the lipid A-treated tumor nodules collected at day
28 (ie, after the last lipid A injection), both the size of the nodules
and the number of tumor cells decreased. Furthermore, the tumor cells
were localized only in the center of the nodules and were surrounded
with an inner crown of fibroblasts and collagen and an outer crown of
lymphocytes (Figure 3B
1). In these nodules, the PROb tumor
cells were the only cells to be stained by the anti-TGF-ß1 antibody,
even though the staining was lighter than in untreated tumors and
involved only half of the PROb cells (Figure 3B
2).
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We have previously shown that the NO-producing cells in tumor
nodules were the PROb tumor cells.20
Constitutively, PROb
cells cultured in vitro produced low amounts of NO (about 6
nmol/ml). The combination of IL-1ß (1 ng/ml) and IFN- (100 U/ml)
for 48 hours significantly increased NO secretion. This effect was
totally inhibited in the presence of the NO synthase inhibitor NMMA
(Figure 4A)
. Addition of 0.1, 1, and 10
µg/ml of lipid A did not stimulate NO secretion by either
unstimulated or cytokine-stimulated PROb cells. At concentrations
ranging from 0.25–10 ng/ml, rhTGF-ß1 did not influence NO production
by either unstimulated or cytokine-stimulated PROb cells (Figure 4A)
,
whereas 0.25 ng/ml TGF-ß1 inhibited NO production of EC 219
endothelial rat cells stimulated with 50 ng/ml TNF- plus 100 U/ml
IFN- (Figure 4B)
. TGF-ß1 remained without effect when added to
cell cultures 24 hours before adding IL-1ß plus IFN- for 48 hours,
as well as when incubation time with the stimulatory cytokines and
TGF-ß1 was increased to 72 and 96 hours or when cytokine
concentrations were increased to 25 ng/ml (IL-1ß) and 250 U/ml
(IFN-), respectively.
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Using CCL64 cells as a positive control, we demonstrated the
expression of TGF-ß type I (RI) and TGF-ß type II (RII) receptors
on PROb cells by Western blotting experiments. Immunoreactive bands
detected in PROb cells migrated at 53 kd and 75 kd with anti-TGF-ß RI
and anti-TGF-ß RII antibodies, respectively (Figure 5A)
. Because no inhibitory effect of
TGF-ß1 had been detected on cultured PROb cells, we performed
cross-linking experiments to study the capacity of these receptors to
bind 125I-TGF-ß1. Whereas two bands were observed for CCL
64 cells, one band corresponding to TGF-ß RI but no band
corresponding to TGF-ß RII was visible for PROb cells (Figure 5B)
.
The same results were obtained in the presence of an anti-TGF-ß RII
neutralizing antibody (not shown). Thus, PROb cells possess a
nonfunctional type II receptor, which precludes normal signal
transduction by exogenous TGF-ß1. These results strongly suggest that
in PROb tumors, the inverse correlation between NO and TGF-ß1 is type
II receptor-independent.
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To check if the decrease in TGF-ß1 concentration observed in
lipid A-treated tumor nodules was the result of the increase in NO
concentration, we measured the total TGF-ß1 concentration in PROb
cells after 48 hours incubation with lipid A, IL-1ß plus IFN-, or
NO donor glyceryl trinitrate (GTN). Figure 6
indicates that the lipid A alone or
combined with the cytokines had no effect on TGF-ß1 synthesis by PROb
cells, nor did GTN at the concentrations tested. The same results were
obtained with another NO donor: the diethylamine NO complex (DEA/NO)
used at same concentrations as GTN. IL-1ß or IFN- alone had no
effect, either.
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Because PROb cells are the NO- and TGF-ß1-secreting cells in
tumor nodules, we hypothesized that endogenous TGF-ß1 could regulate
NO secretion by PROb cells. To determine the role of endogenous
TGF-ß1, PROb cells were transfected with an expression vector coding
for TGF-ß1 antisense mRNA. As demonstrated in Figure 7A
, the transfection successfully
decreased the TGF-ß1 secretion of PROb cells but did not modify their
TGF-ß RI and TGF-ß RII expression (not shown). The TGF-ß1
production of the uncloned transfected PROb cells (PROb Bulk) was
0.009 ± 0.003 ng/mg protein, compared to 0.018 ± 0.002
ng/mg protein for the nontransfected cells or 0.017 ± 0.005 ng/mg
protein for the PROb cells transfected with the vector alone (PROb
Neo). Among the different clones obtained from the PROb Bulk cells, two
clones were selected: clone 3', which secreted a comparable amount of
TGF-ß1 as the parental PROb cells (0.016 ± 0.004 ng/mg
protein), and clone 13, which secreted barely detectable levels of
TGF-ß1 (0.004 ± 0.003 ng/mg protein).
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in a medium without FBS.
Figure 7B
shows that there was an inverse correlation between the
TGF-ß1 secretion of the different PROb cell clones and their ability
to secrete NO when stimulated. The PROb Bulk and the clone 13 cells
secreted significantly (P 
0.001) more NO when
compared to the nontransfected PROb cells, the transfected clone 3',
and the PROb Neo cells. The NO secretion of the PROb Bulk and clone 13
cells was more than twice as high as that of the control PROb cells.
This production was inhibited in the presence of NMMA, which had no
effect by itself. Exogenous TGF-ß1 (0.5, 1, or 5 ng/ml) had no effect
on NO secretion by the four PROb cell clones. TGF-ß1 Acted as an Intracellular Mediator for NO Synthesis Inhibition in PROb Cells
To study the mechanism by which TGF-ß1 down-regulates NO
secretion in PROb cells, we measured NO synthesis of the different PROb
cell clones under stimulation with IL-1ß plus IFN- in the presence
or in the absence of an anti-TGF-ß RII neutralizing antibody.
Because no commercial anti-rat TGF-ß RII antibody was available, we
used a human antibody to block TGF-ß RII on PROb cells. To be sure of
TGF-ß RII inhibition, we first tested the cross-reactivity of the
antibody on rat cells. Figure 4B
shows that the rat endothelial EC 219
cells secreted NO when stimulated with TNF- plus IFN-; this
secretion was partially inhibited by 0.25 ng/ml TGF-ß1. The addition
of 20 µg/ml of the anti-human TGF-ß RII antibody inhibited 50% of
the TGF-ß1 effect on NO synthesis by EC 219 rat cells, whereas the
addition of 20 µg/ml of an unrelated human antibody had no effect.
Since the parental PROb cells secrete about 0.018 ng/ml TGF-ß1
(Figures 6 and 7A)
, 20 µg/ml of the anti-TGF-ß RII antibody should
completely neutralize a potential autocrine effect of exogenous
TGF-ß1, supposing that the TGF-ß RII is somehow functional despite
the cross-linking data to the contrary.
Figure 8
indicates that in the presence
of the anti-TGF-ß RII antibody the basal and induced production of NO
of the different PROb cell clones were not modified. Thus, in PROb
cells, TGF-ß1 appears to inhibit NO synthesis by an intracellular
mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated the strong antitumor efficiency of a lipid A
immunotherapy in the syngeneic model of peritoneal carcinomatosis
induced by PROb colon carcinoma cells in BDIX rats. In
vitro, lipid A shows neither antiproliferative nor cytotoxic
activity on PROb cells. In vivo, tumor regression induced by
the lipid A therapy is associated with an increased NO content in tumor
nodules.20
The present study demonstrates that constitutive
NOS is not expressed in tumor nodules and that the increased NO content
identified in tumor nodules regressing upon lipid A therapy is related
to the accumulation of iNOS. In PROb tumors, the PROb tumor cells were
identified as the cells responsible for NO production. Cultured PROb
cells do not spontaneously secrete NO and lipid A has no direct effect
on this production, but the combination of IL-1ß and IFN-
stimulates NO production by PROb cells in vitro. In
vivo immunohistological studies showed an accumulation of IL-1ß
and IFN- in lipid A-treated tumor nodules compared to untreated
nodules, suggesting a possible role for these cytokines in the
increased intratumoral level of iNOS and NO in treated
rats.20
NO can be cytotoxic for tumor cells in
vitro and demonstrates antitumor properties in
vivo.14,17,18
In vitro, we showed that NO
secreted by PROb cells under IL-1ß plus IFN- stimulation inhibits
their proliferation.20
Therefore, an increased NO
intratumoral concentration appears to account for tumor regression
observed in lipid A-treated animals. Nevertheless, until now, we had
not been able to demonstrate this clearly, because the administration
of NMMA or PROb cell transfection with an expression vector encoding
for iNOS antisense mRNA did not allow a decision on this point.
TGF-ß1 has been shown to inhibit NO production by a variety of normal
cells5,11,12,27,28
and TGF-ß1 null mice demonstrated NO
serum levels fourfold higher than control animals.29
To our
knowledge, the relationships between TGF-ß1 and NO synthesis had
never been analyzed in tumor cells. In PROb tumor nodules, we show that
the intratumoral concentration of TGF-ß1 is high during the growth of
tumor nodules and decreases in nodules whose regression is induced by
the lipid A therapy. Like many other colon cancer cell
lines,6,8
cultured PROb cells secrete
TGF-ß1.2,3,7
In subcutaneous PROb tumors developed in
rats7
as well as in human colon tumors,4
TGF-ß1 was shown to be produced exclusively by tumor cells.
Immunohistological studies performed on peritoneal PROb tumor nodules
from either untreated or lipid A-treated rats confirmed that tumor
cells were the only cells to be stained with the anti-TGF-ß1
antibody. While nearly all the tumor cells were stained in tumor
nodules from untreated rats, only half of them were stained in those
from lipid A-treated animals. Craven et al30
showed that NO
generated exogenously by the NO donor
S-nitroso-N-acetyl,D,L,-penicillamine or endogenously after exposure of
mesangial cells to IL-1ß suppressed bioactive TGF-ß in these cells.
In PROb cells, the lipid A alone or in combination with IL-1ß and
IFN- has no effect on TGF-ß1 synthesis, nor have the NO donors GTN
and DEA/NO. Therefore, the decreased TGF-ß1 concentration observed in
tumor nodules regressing upon lipid A therapy was not due to NO and
might involve other cytokines secondarily induced in vivo.
Thus, in peritoneal PROb tumor nodules, both NO and TGF-ß1 were
secreted by tumor cells but NO content was inversely related to
TGF-ß1 content during tumor growth and regression induced by the
lipid A.
TGF-ß1 is known to interact with cell surface receptors expressed on
the plasma membrane of target cells.6,8,25,31
Based on
radioimmunoassay data, PROb cells bind TGF-ß1.2
The
present Western blotting study shows that they express both type I and
type II receptors, so that the inverse relationship between TGF-ß1
and NO levels in PROb tumor nodules could have been explained if
TGF-ß1 secreted by tumor cells modulated NO synthesis in these cells
by an autocrine or paracrine mechanism. In fact, exogenous TGF-ß1 did
not influence NO synthesis by cultured PROb cells, either unstimulated
or stimulated by various combinations of IL-1ß plus IFN-.
Exogenous TGF-ß1 did not influence PROb cell proliferation,
either.3
A similar loss of responsiveness to exogenous
TGF-ß1 was reported in some colon cancer cell lines as well as in
advanced human colon cancers.6,31
Three major types of
TGF-ß-binding proteins have been identified. TGF-ß RIII may be
involved in ligand presentation to TGF-ß signaling
receptor.8,31
whereas molecular cloning and functional
analyses have shown that both RI and RII receptors, are necessary for
TGF-ß signal transduction.6,8,25,31
Here, cross-linking
data indicated that the absence of any effect of exogenous TGF-ß1 on
PROb cells correlates with the fact that TGF-ß RII does not bind
125I-TGF-ß1, whereas RI does. A growing body of evidence
suggests that some TGF-ß1 biological effects would be type II
receptor-dependent, whereas others would be type II
receptor-independent. In a subclone of the MCF-7 cell line,
cross-linking experiments showed that the TGF-ß RII was not
functional, yet the TGF-ß RI remained capable of binding TGF-ß1.
The inactivation of the TGF-ß RII has been shown to induce a specific
abrogation of the TGF-ß1 antiproliferative effect without affecting
the TGF-ß1 stimulation of its own transcription32
or of
the extracellular matrix.33
TGF-ß1 is known to be secreted by most cells in an inactive (latent)
form but PROb cells, like several other colon tumor cell
lines,6
were shown to secrete a fraction of bioactive
TGF-ß1.2
As the anti-TGF-ß1 antibody used here for
immunological studies specifically recognized active TGF-ß1, PROb
cells might contain intracellular active TGF-ß1 as reported inside B
cells and plasma cells.34
A growing body of evidence
indicates that some cytokines can act not only via cell surface
receptors, but also within the cell.35-38
Because the PROb
cells were both the NO- and TGF-ß1-secreting cells in tumors, we
hypothesize that endogenous TGF-ß1 could negatively regulate NO
secretion in these cells. Transfection of PROb cells with an expression
vector encoding TGF-ß1 antisense mRNA significantly decreased their
TGF-ß1 synthesis. The absence of effect of exogenous TGF-ß1 and the
increased NO secretion in cytokine-stimulated PROb transfected cells,
even in the presence of an anti-TGF-ß RII neutralizing antibody,
strongly argue for an intracellular effect of TGF-ß1 on NO synthesis
down-regulation in PROb tumor cells. Using microinjection technology,
Smith et al demonstrated an intracellular role for IFN- and
TNF-.35
Thus, an intracellular action of some cytokines
may be fairly common.
Nuclear localization signals have been identified in several cytokines
or their receptors.36,38
Some cytokine-receptor complexes
have been localized in the nucleus36,37
but the role of
these complexes within the nucleus is unknown. They could act as
chaperones to accomplish a cytokine-specific nuclear translocation of
signaling molecules that function within the nucleus.36
Some of these complexes were demonstrated to have a DNA binding
capacity, suggesting their implication in modulation of gene
transcription.37
In cell systems responding to exogenous
TGF-ß1, down-regulation of NO synthesis can occur at the
transcriptional, posttranscriptional, and/or posttranslational
level.5,11,12
As PROb cells synthesize TGF-ß RI able to
bind TGF-ß1, such complexes might act as chaperones for SMAD
complexes, recently shown to be involved in TGF-ß signaling and in
the pathogenesis of colorectal cancer.39
Nuclear
factor-
B (NF-B) is implicated in the regulation of the iNOS gene
transcription.40
Exogenous TGF-ß1 was shown to inhibit
NF-B activity by increasing the transcription of I-B, a
protein inhibitor of NF-B.41
It is possible that
endogenous TGF-ß1 linked to TGF-ß RI increases this transcription.
At the present time, endogenous TGF-ß1-mediated regulation of iNOS
expression remains to be fully explained.
To summarize, cytokines may exert their effect not only through surface receptor-driven events, but also through interactions with intracellular targets distinct from surface receptors. Our study supports for the first time an intracellular action of TGF-ß1.
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Acknowledgements |
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Supported by research grants from the Conseil Regional de Bourgogne, OM Laboratories, the Ligue contre le Cancer de l'Yonne, de la Côte d'Or et de la Haute Marne, the Fondation Singer-Polignac, the Fondation de France, and l'Association pour la Recherche contre le Cancer (A.R.C.).
Accepted for publication February 19, 1999.
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production. Role of tumor-derived IL-10, TGF-ß, and prostaglandin E2. J Immunol 1994, 153:1674-1686[Abstract]
. Microinjection of human IFN- induces Ia expression on murine macrophages. J Immunol 1990, 144:1777-1782[Abstract]
B/REL proteins. Biochem Biophys Res Commun 1995, 209:73-79[Medline]
B/REL activity inducing apoptosis of B cells: transcriptional activation of IB. Immunity 1996, 5:31-40[Medline]
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) or not
(
) with OM 174. Tumor
nodules were taken from rats of both groups on days 15, 22, 29, 35, and
42 after the tumor cell injection, homogenized in an extraction buffer,
and centrifuged. The total TGF-ß1 concentration of the supernatants
was measured by the ELISA method. The statistical analysis was
performed using Student's t-test
(n.s., not significant; **, P 
