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Clinical Cancer Research Vol. 9, 4562-4569, October 1, 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Is Interferon {gamma} One Key of Metastatic Potential Increase in Human Bladder Carcinoma?

Pierre Champelovier1, Annick Simon, Catherine Garrel, Geraldine Levacher, Vincent Praloran and Daniel Seigneurin

Laboratoire de Cytologie [P. C., D. S.], Laboratoire d’Hématologie [A. S.], both from Département de Biologie et de Pathologie de la Cellule, and Laboratoire de Biologie du Stress Oxydatif, Département de Biologie Intégrée [C. G.], Centre Hospitalier Universitaire de Grenoble, 38043 Grenoble Cedex; Laboratoire Migration Cellulaire et Infiltration Tumorale, Institut Albert Bonniot, La Tronche [G. L.]; and FRE 2617 du Centre National de la Recherché Scientifique, Université de Bordeaux 2, 33076 Bordeaux Cedex [V. P.], France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 B. Effects of Low...
 DISCUSSION
 REFERENCES
 
Purpose: IFN-{gamma} is detected in the urine of bladder cancer patients after intravesical bacillus Calmette Guerin instillation. Because it acts in the anticancer process, we studied its cellular and molecular mechanisms of action on human bladder cancer cell lines.

Results: IFN-{gamma} (>5 ng·ml-1)(>400 IU·ml-1) inhibited the growth of bladder cancer cell lines and modified the expression of the tumor-associated markers tissue-type plasminogen activators, Plasminogen activator inhibitor-2, urokinase plasminogen activator receptor, colony-stimulating factor 1, intercellular adhesion molecule 1, and class II MHC. Interestingly, IFN-{gamma}-induced apoptosis of the low-grade bladder cancer cell lines (RT4/G1 and RT112/G2) related to a cleavage of caspases 1, 8, and 9. This process was inhibited by the phosphatidylinositol 3'-kinase inhibitor (LY294002) and the protein synthesis inhibitor (cycloheximide). Moreover, low doses of IFN-{gamma} (<5 ng·ml-1)(<400 IU·ml-1) increased the resistance to the cytotoxic effect of tumor necrosis factor {alpha} in the RT112 cells but not in the RT4 cells. This acquired resistance was associated with morphological changes and with an increase of the cell migration and scattering.

Conclusions: We demonstrated that in the low-grade bladder cancer cell lines, the effect of IFN-{gamma} was dose dependent: high doses (>5 ng·ml-1) induced apoptosis of RT4 and RT112 cells, whereas low doses (<5 ng·ml-1) induced a resistance to the cytotoxic effect of tumor necrosis factor {alpha} and increase the metastatic potential of the RT112 cells. Therefore, we propose that a similar phenomenon could participate to the immunotherapy failure observed during tumor progression of bladder cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 B. Effects of Low...
 DISCUSSION
 REFERENCES
 
Bladder cancer patients are cured by transurethral resection associated with immunotherapy (intravesical BCG2 therapy). Several authors have confirmed the efficacy of the BCG therapy first described by Morales et al. (1) in the treatment of 90% of superficial bladder carcinomas (2) with 10% of patients showing tumor progression (muscle invasion and metastasis). Local intravesical BCG immunostimulation is characterized by a major infiltration of T lymphocytes (helper/CD4+) in bladder wall and secretion of IL-1{alpha}/ß, IL-2, IL-5, IL-6, IL-8, IL-10, TNF-{alpha}/ß, IFN-{gamma} and CSF-1 (3, 4, 5, 6) mostly produced by macrophages, lymphocytes, endothelial cells, and fibroblasts infiltrating the tumor but also by urothelial tumor cells, suggesting that the tumor itself could play a role in the mode of action of BCG (7 , 8) .

IFN-{gamma} is a pleiotropic immunomodulatory lymphokine that enhances class II MHC (HLA-DR), cell surface antigens (uPA-R), ICAM-1/CD54, intracellular proteins (PAI1 and PAI2), and several growth factors (HGF, TNF-{alpha}, and CSF-1; Refs. 9, 10, 11 ). Some of these phenotypic markers are associated with a poor outcome of cancer patients (12, 13, 14, 15, 16) . In addition, in vitro treatment of tumor cells with IFN-{gamma} decreased their sensitivity to cytotoxic agents and increased experimental metastasis (17 , 18) .

This work investigated the cellular and molecular mechanisms by which IFN-{gamma} affects urothelial tumor cell growth, apoptosis, tumoral potential, and invasion activity of six bladder cancer cell lines (RT4, RT112, DAG-1, T24, J82S, and TCCsup). Our results demonstrated that high doses (5–50 ng·ml-1/400-4000 IU·ml-1) of IFN-{gamma} induced apoptosis of the low-grade bladder cancer cell lines (RT4 and RT112), whereas low doses (0.05–5 ng·ml-1/4–400 IU·ml-1) induced the resistance to the cytotoxic effect of TNF-{alpha} and increased the tumoral potential (scattering and cell motility) of the RT112 cells. IFN-{gamma} did not modify phenotype of the high-grade bladder cancer cell lines (morphology, apoptosis, and migration). These results suggest that the effects of IFN-{gamma} on bladder cancer cells is very complex and likely to be cell dependent.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 B. Effects of Low...
 DISCUSSION
 REFERENCES
 
Maintenance of Human Urothelial Cell Lines.
Six bladder carcinoma cell lines (RT4/G1 grade, RT112/G2, DAG-1/G3, T24/G3, J82S/G3, and TCCsup/G4) were used in our experiments. Their grade (G1, G2, G3, and G4) was defined according to the grade of the original tumor. These adherent human urothelial cells were maintained in RPMI 1640 with 10% (v/v) inactivated FCS (Life Technologies, Inc., Eragny, France), antibiotics (penicillin 100 IU·ml-1 and streptomycin 100 µg·ml-1) and L-glutamine (2 mM; Roche, Meylan, France).

Expression and Production of Cytokines and Other Molecules.
Cells were grown in RPMI 1640 with 1% FCS in presence of 1–50 ng·ml-1 IFN-{gamma} for 8–96 h. The 48 h CSs were centrifuged (10 min, 200 x g) to remove cells and debris, then stored at -80°C until analysis of the CSF-1, PAs, PAIs, HGF, or TNF-{alpha} antigen levels. PAs and PAIs (Biopool), TNF-{alpha} (Bechman-Coulter), and HGF (R&D Systems, Minneapolis, MN) were determined with ELISA according to the manufacturer’s instructions. A specific ELISA developed in one of our laboratories was used to measure CSF-1 levels (19) . Nonadherent and adherent trypsinized cells were used for the determination of cell proliferation, cell death (apoptosis) and antigen expression of u-PAR/CD87, PAI2, ICAM-1/CD54, and HLA-DR using flow cytometry. MnSOD, catalase, and GPX were determined as described previously (20) . Membrane associated antigens were analyzed by indirect immunofluorescence using specific monoclonal antibodies. Cytoplasmic immunophenotyping was determined after cell permeabilization (IntraPrep Permeabilization Reagent/Bechman-Coulter, Marseille, France). Specific conjugated secondary antibodies and appropriate conjugated isotypic controls were added before measuring antigen expression by their fluorescence intensity by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA).

Drugs and Reagents.
Recombinant human IFN-{gamma} and TNF-{alpha} (R&D Systems) were diluted to appropriate final concentrations in RPMI 1640. Mouse monoclonal antibody anti-PAI2 (MAI21) was purchased from Biopool (Umeä, Sweden), anti-u-PAR/CD87 from American Diagnostic (Greenwich, CT), anti-ICAM-1/CD54 and anti-HLA-DR were from Bechman-Coulter. Mouse monoclonal antibodies anti-TNF receptor I (TNF-RI/p55/CD120a) and anti-TNF receptor II (TNF-RII/p75/CD120b) were kindly provided by Manfred Brochkaus (F. Hoffman-La Roche, Basel, Switzerland), whereas anti-IFN-{gamma} receptor (IFN{gamma}-R/CD119) was purchased from Caltag (Burlingame, CA). Polyclonal antibodies anti-Akt and anti-Akt-p and monoclonal antibody anti-PTEN were purchased from Cell Signaling Technology (New England Biolabs, Hitchin, United Kingdom), polyclonal anti-IAP2 and monoclonal anti-BclxL antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anti-TNF-{alpha} antibodies (neutralizing) were from Genzyme (Cambridge, MA). IL-1ß inhibitor-II (inhibitor of caspase 1), AcD (inhibitor of RNAs synthesis), CHX (inhibitor of proteins synthesis), PDTC (inhibitors of nuclear factor-{kappa}B), staurosporine (inhibitor of protein kinase C), genistein (inhibitor of tyrosine protein kinase), H7 (inhibitor of PKA), LY294002 (inhibitor of PI3k), SB (inhibitor of p38 MAPK), and PD (inhibitor of ERK) were purchased from Sigma (St. Louis, MO) and used as specified by the manufacturer.

Transwell Motility and Cell Migration Assays.
It was carried out using Transwell chamber equipped with an uncoated polycarbonate filter membrane (8-µm pores; Corning Costar, Cambridge, MA). Tumor cells were plated at 105 cells·cm-2 in the upper compartment of the chamber and cultured for 24 h. Motility, expressed as a percentage, was the number of cells that moved through the filter divided by the total number of cells in the chamber x 100. Cell migration was assessed using a scratch wound assay. Cells cultured in RPMI 1640 with 1% FCS with or without IFN-{gamma} to nearly confluent cell monolayers were then wounded with sterile pipette. The cellular debris were removed, and the cells grown for 72 h then photographed under a phase contrast microscope.

Assessment of Proliferation and Apoptosis.
Total and viable cell number was determined using trypan blue. The flow cytometry analysis of cell DNA content (CycleTest PLUS/DNA reagent kit; Becton Dickinson) allowed to determine the cycle phase of tumor cells before and after treatment together with those undergoing apoptosis (hypodiploid). Apoptosis was confirmed using Annexin V-FITC/PI method (Vybrant Apoptosis Assay kit; Molecular Probes, Eugene, OR).

Statistical Analysis.
For all variables included in the present study, their mean values, SD, and ranges were calculated using the Statview software package (Macintosh). The statistical significance was P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
B. Effects of Low...
 DISCUSSION
 REFERENCES
 
A. Effects of high doses of IFN-{gamma} (5–50 ng·ml-1)
Growth Inhibition.
A high dose of IFN-{gamma} (20 ng·ml-1) inhibited the growth of all bladder cancer cell lines (25 ± 4% after 96 h; P = 0.038) and reduced the percentage of cells in S phase (12 ± 3 versus 20 ± 2% in control culture). Interestingly, IFN-{gamma} also decreased the absolute cell number only of low-grade bladder cancer cell lines (RT4/G1 and RT112/G2), suggesting a cytotoxic effect.

Modulation of the Cell Phenotype.
In the six bladder cancer cell lines, IFN-{gamma} increased the expression of several tumor-associated markers (ICAM-1, PAI2, t-PA, uPA-R, HLA-DR, CSF-1) but not others (uPA, PAI1; Table 1Citation ). Moreover, RT112 but not RT4 cells acquired a fibroblast-like morphology after 24 h of culture in presence of IFN-{gamma}. The constitutive fibroblast-like morphology of high grade bladder cell lines (DAG-1, T24, J82S, and TCCsup) was not modified by IFN-{gamma}.


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  		Table 1 Phenotype changes of IFN{gamma}-induced BCCL

 
Apoptosis.
When treated with 20 ng·ml-1 IFN-{gamma}, RT4 and RT112 adherent cells progressively detached and showed signs of apoptosis (cytoplasmic vacuolation, nucleoli disappearance, and nuclei condensation; Fig. 1Citation ). When compared with cultures without IFN-{gamma} (DNA cycle test kit/Fig. 2ACitation ), flow cytometry showed that IFN-{gamma} increased the percentage of apoptotic cells (subdiploid) that was confirmed by Annexin V-FITC/PI test (Fig. 2B)Citation . The percentage of apoptotic cells correlated inversely with the cytological grade of the tumor cells: it decreased from RT4/G1 (31 ± 3%) to TCC/G4 (<4%).



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  		Fig. 1. Morphology of induced cells. RT4 (top) and RT112 (bottom) cells (5 x 105 cells/ml) grown for 48 h in RPMI 1640 1% FCS without (left) or with 20 ng/ml IFN-{gamma} (right). Cultures were then trypsinized, cells cytocentrifuged, and the slides stained using May Grunwald Giemsa. Arrows indicate apoptotic cells.

 


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  		Fig. 2. Flow cytometry analysis of apoptosis. RT4 and RT112 cells (5 x 105 cells/ml) were cultured without (T) or with 20 ng/ml IFN-{gamma} for 48 h. Cultures were then trypsinized, cells stained using either DNA cycle test kit (A) or Annexin V-FITC/PI kit (B) before flow cytometry analysis (FACScalibur). A, arrows indicate subdiploid population (sub-G1); B, numbers indicate the percentage of apoptotic cells.

 
To investigate apoptosis signaling pathway, several inhibitors were added 4 h before the addition of IFN-{gamma}. The p38 MAPK inhibitor (SB) and the PKA inhibitor (H7) both increased IFN-{gamma}-induced apoptosis, whereas the PI3k inhibitor (LY294002), the protein synthesis inhibitor (CHX), and PDTC that prevents the signal-induced phosphorylation of inhibitor of nuclear factor-{kappa}B{alpha} inhibited it (Table 2)Citation . The tyrosine protein kinase inhibitor (genistein), the mRNA synthesis inhibitor, (AcD), the MAP/extracellular signal-regulated kinase inhibitor (PD098059), and the protein kinase C inhibitor (staurosporine) had no effect.


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  		Table 2 Effect of transduction pathway inhibitors on RT112 TNF-{alpha}- and IFN-{gamma}-induced apoptosisa

 
IFN-{gamma} have been reported to have pleiotropic effects, including ability to induce the production of other cytokines in several cell types (21) . Twenty ng·ml-1 of IFN-{gamma} for 48 h was ineffective on TNF-{alpha} production by RT4 cells, whereas it induced low levels of TNF-{alpha} antigen that increased from 0.25 to 0.50 ng·ml-1 (for 106 cells and 48-h induction time). Moreover, in RT112 cells, polyclonal neutralizing anti-TNF-{alpha} antibodies specifically decreased the IFN-{gamma}-induced apoptosis, suggesting a role for induced TNF-{alpha} (Table 3)Citation .


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  		Table 3 Effect of anti-TNF-{alpha} antibodies on IFN-{gamma}-induced apoptosis

RT4 and RT112 cells were treated with specific anti-TNF{alpha} (100 µg/ml) or with irrelevant IgG (100 µg/ml). Four h later, cells were treated without (control) or with 20 ng/ml IFN-{gamma} for 48 h. Apoptosis was measured with Annexin V/FITC kit. Results are the mean ± SD of two experiments.

 
The cleavage of caspases (a family of cysteine proteases) may be critical in apoptosis. A cleaved form of caspases 8 and 9 appeared after 24 h of IFN-{gamma} treatment (Fig. 3A)Citation as evidenced by Western blotting analysis with specific antibodies. Caspase-1 inhibitor (ICEi-II) reduced the cytotoxic effect of IFN-{gamma} in a dose-dependent manner in RT4 and RT112 cells, suggesting that in IFN-{gamma}-sensitive bladder tumor cells, apoptosis is mediated by induction of caspase 1 (Fig. 3B)Citation . High doses of ICE inhibitor (100 µM) also inhibited IFN-{gamma}-induced RT112 cell morphological changes. ICE inhibitor did not modify the TNF-{alpha}-induced apoptosis. IAP protein family has been implicated in signal transduction program in TNF-{alpha}-induced apoptosis. IFN-{gamma} did not modify IAP2 expression in both RT4 and RT112 cells. In contrast, TNF-{alpha} decreased it in RT112 but not in RT4 cells, a phenomenon probably related to the low percentage of TNF-{alpha}-induced apoptotic cells in the RT4 cell line (<10%; Fig. 3CCitation ). In both RT4 and RT112 cell cultures treated with TNF-{alpha} + IFN-{gamma}, the percentage of apoptotic cells increased dramatically (synergistic effect) in relation with a strong decrease of IAP2 and Bclxl antigen expression (Fig. 3C)Citation . By contrast TNF-{alpha} + IFN-{gamma} did not modify either IAP2 or Bclxl antigen expression and did not induced apoptosis in high-grade bladder cell lines (data not shown).



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  		Fig. 3. Caspases, Bclxl, and IAP2 antigen expression. A, RT112 cells (5 x 105 cells/ml) were treated with 20 ng/ml IFN-{gamma} for 24 h, before lysis, SDS-PAGE, and Western blotting using anticaspase 8 and anticaspase 9 antibodies. C-, negative control (noncleaved caspase); C+, positive control (noncleaved and cleaved caspase); T, noninduced cells; TNF-{alpha}, TNF{alpha}-induced cells; IFN-{gamma}, IFN-{gamma}-induced cells. B, RT112 cells were cultured without or with IL-1ß converting enzyme inhibitor-II (ICEi-II) for 4 h then treated without inducer (T) or with TNF-{alpha} (30 ng/ml) or IFN-{gamma} (30 ng/ml) for 48 h. Apoptosis was determined using Annexin-V/FITC technique. Results are the mean ± SD of two experiments. C, RT4 and RT112 cells (5 x 105 cells/ml) were treated for 24 h without or with TNF-{alpha} (20 ng/ml), IFN-{gamma} (20 ng/ml), or both TNF-{alpha} and IFN-{gamma}. Cells were then treated as described in A, and Western blot was done using polyclonal anti-IAP2 and monoclonal anti-Bclxl antibodies. The percentage of apoptotic cells was determined using Annexin V-FITC/PI technique. Legend: as described in A.

 

   B. Effects of Low Doses of IFN-{gamma} (0.05–5 ng·ml-1/4–400 IU·ml-1)
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 B. Effects of Low...
DISCUSSION
 REFERENCES
 
Acquired Resistance to the Cytotoxic Effect of TNF-{alpha}.
A low-dose of IFN-{gamma} did not affect the growth of all of the six bladder cancer cell lines but induced a slight apoptotic process in both the RT4 and RT112 cell lines. Moreover, when RT112 cells were treated for 48 h with low doses of IFN{gamma} (<2 ng·ml-1) before addition of TNF-{alpha} (30 ng·ml-1 for 48 h), the percentage of apoptotic cells decreased (24 ± 2 versus 41 ± 3% in IFN-{gamma}-treated and control cultures, respectively; Fig. 4Citation ).



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  		Fig. 4. IFN-{gamma}-induced chemoresistance. RT112 cells (5 x 105 cells/ml) were cultured without (A) or with IFN-{gamma} (5 ng/ml) for 48 h (B; pretreatment). Cells were then washed twice in RPMI 1640 and induced with TNF-{alpha} (30 ng/ml) for 48 h (C and D). Cultures were then trypsinized and cells stained using DNA cycle test kit before flow cytometry analysis.

 
This effect is probably related to IFN-{gamma}-induced protein synthesis because it is observed only after 24 h of preculture. Incubation of RT112 cells with CHX, a protein synthesis inhibitor, 4 h before the addition of IFN-{gamma} inhibited dramatically IFN-{gamma}-induced resistance to the cytotoxic effect of TNF-{alpha} (Table 4)Citation . Antioxidant enzymes (catalase, MnSOD, and GPX) protect cells against apoptosis (22) . IFN-{gamma} significantly increased MnSOD (3 ± 0.04 versus 2.05 ± 0.03 units/mg) and GPX (0.33 ± 0.04 versus 0.18 ± 0.03 units/mg) activity when compared with the control. However, IFN-{gamma} increased MnSOD and GPX of both RT4 cells (that did not acquire resistance) and all of the constitutively resistant high-grade bladder cell lines (DAG-1, T24, J82S, and TCCsup). Therefore, alone, IFN-{gamma}-induced antioxidant activity did not explain the resistance of bladder cancer cells to the cytotoxic effect of TNF-{alpha}.


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  		Table 4 IFN-{gamma}-induced chemoresistance in RT112: effect of transduction pathway inhibitors

RT112 cells, growing in RPMI 1640 with 1% FCS, were treated without or with inhibitor (AcD, CHX, LY, SB, or PD) for 4 h then treated without (-) or with 5 ng/ml IFN{gamma} (+) for 48 h. Cells were washed twice and recultured in presence of TNF-{alpha} (30 ng/ml) for 24 h. Then, apoptosis was determined using DNA cycle test kit. Morphology was estimated using inverted microscope after washing. (R, normal round phenotype; F, fibroblast-like phenotype). Results are the mean ± SD of three experiments.

 
Cell Signaling Pathway.
Akt activation (phosphorylated-Akt/Akt-p) protects cells from apoptosis, whereas frequent loss of PTEN expression (linked to elevated phosphorylated-Akt levels) is associated with metastatic process (23) . Therefore, using antibodies specific for PTEN and for the phosphorylated serine 473 of Akt (Akt-p), we examined if IFN-{gamma} treatment sustained modulation of both Akt-p and PTEN antigen expression. In the IFN-{gamma}-induced RT112 cells, increase of phosphorylated-Akt antigen levels was detected between 4 and 6 h after stimulation. The mean fluorescence intensity as determined using fluorescence-activated cell sorting analysis increased from 30 ± 5 in the control cells to 120 ± 8 after 4 h of IFN-{gamma} treatment (x4). The PI3k inhibitor, LY294002, blocked the IFN-{gamma}-induced protective effect, confirming a role for Akt, whereas the MAP/extracellular signal-regulated kinase inhibitor PD098059 or the p38/MAPK inhibitor SB did not. However, this latter inhibitor blocked the IFN-{gamma}-induced morphological changes. PDTC partially inhibited chemoresistance and inhibited morphological changes IFN-{gamma} induced (Table 4)Citation . All of the six bladder cancer cell lines expressed high PTEN antigen levels without correlation with the constitutive or acquired chemoresistance (P < 0.5).

Cell Death Receptor Expression.
TNF-{alpha} induces an apoptotic process through a specific receptor (TNF-RI/p55). Using a specific monoclonal antibody (CD120a) directed against the TNF-RI/p55, we showed that IFN-{gamma} increased its expression (52 ± 7 versus 30 ± 3% in IFN-{gamma}-treated and control culture, respectively). This result suggested that IFN-{gamma}-induced chemoresistance is independently regulated by the cell death receptor expression.

HGF Secretion.
IFN-{gamma} also modified the morphology of the cells that exhibited extensive branching and induced scattering (Fig. 5Citation , top). HGF/scatter factor induces resistance to the cytotoxic effect of TNF-{alpha}, and/or morphological change (24) was then determined in CSs by ELISA. In the RT112 cells, IFN-{gamma} increased HGF antigen levels (15 versus <5 ng·l-1) in induced and control culture for 106 cells and 48-h induction time. AcD inhibited the secretion of HGF, the morphological change, and the resistance to the cytotoxic effect of TNF-{alpha}, suggesting that IFN-{gamma} induces HGF at the mRNA levels. The IFN-{gamma}-induced biological effects were partially blocked by using specific anti-HGF polyclonal antibodies. The percentage of RT112 cells possessing a fibroblast-like morphology decreased from 30 ± 5 to 12 ± 4%). Exogeneously added HGF (CS from MRC5 cells) induced both the dissociation of RT112 cell aggregates and the resistance to the cytotoxic effect of TNF-{alpha}. Moreover, DAG-1, J82S, and TCCsup cells that secreted high levels of HGF (25 ± 2, 20 ± 3, and 26 ± 5 ng·l-1 for 106 cells and 48-h induction time, respectively) expressed a constitutive resistance to the cytotoxic effect of TNF-{alpha} and had a fibroblast-like morphology. Their CS also induced resistance to the cytotoxic effect of TNF-{alpha} and scattering of the RT112 cells (data not shown). IFN-{gamma} did not induce either morphological change or HGF secretion of the RT4 cells. However, CS from MRC5 cells slightly modified RT4 cell morphology in a dose-dependent fashion. Therefore, all these results suggest that in the bladder tumor cells, HGF may have an important role in the IFN-{gamma}-induced resistance to the cytotoxic effect of TNF-{alpha} and the morphological change through an Akt signaling pathway.



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  		Fig. 5. Effect of IFN-{gamma} on the RT112 cell morphology and migration. RT112 cells (5 x 105 cells/ml) were grown in RPMI 1640 without (left) or with 5 ng/ml IFN{gamma} (right). After 48 h of culture, confluent cultures were scratch wounded using sterile pipette (T = 0 h). After 72 h, cultures were photographed under a phase contrast microscope (bottom). [———], scratch limit.

 
Cell Migration.
Because the ability of the tumor cells to migrate has been associated to the metastatic potentiality, the cell migration of IFN-{gamma}-treated cells was investigated. Confluent RT112 cells were scratch wounded and recultured for 72 h. Cells spreading along the edges of the wound was significantly faster in IFN-{gamma}-induced than in control cells (Fig. 5Citation , bottom). These results were confirmed by the cell migration assay. IFN-{gamma}-induced RT112 cells migrate through membrane, whereas control cells did not (10 versus 0.3%). The cell migration of the IFN-{gamma}-treated RT4 cells was not modified (0.4 versus 0.3%).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 B. Effects of Low...
 DISCUSSION
REFERENCES
 
Clinical trials have confirmed that BCG therapy exerts antitumor effects by activating effector cells that produce antiproliferative cytokines and by inhibiting tumor growth directly (8 , 25) . On the basis of these results, the in vitro effects of IFN-{gamma} (a BCG therapy-related cytokine) was studied on six bladder cancer cell lines. Cell proliferation, apoptosis, cell differentiation, resistance to the cytotoxic effect of TNF-{alpha}, cell motility, and morphological changes were investigated with 0.05–50 ng·ml-1 IFN-{gamma}.

High doses of IFN{gamma} (>5 ng·ml-1) (>400 IU/ml) inhibited cell proliferation of all of the six bladder cancer cell lines. They also modulate cell phenotype by increasing expression of tumor-associated markers (t-PA, uPA-R/CD87, PAI2, CSF-1, ICAM-1/CD54, HGF, and HLA-DR), all expressed at a high level in the G3/G4 grade bladder cancer cell lines and associated either with invasiveness or with poor prognosis in numerous epithelial cancers (13, 14, 15, 16 , 26, 27, 28) . However, several authors showed that this tumor-associated proteins may contribute to tumor regression in vivo (29 , 30) . High doses of IFN-{gamma} were also able to induce apoptosis of the low-grade bladder cancer cell lines in a time-dependent manner. Moreover, Shin et al. (31) showed that IFN-{gamma} was able to increase MMP-9 in several bladder tumor cell lines, suggesting that it may enhance the invasiveness of the tumor. However, Slaton et al. (32) showed that treatment of bladder tumor with IFN-{alpha} associated with chemotherapy restores the balance between MMP-9 and E-cadherin, suggesting that IFN may limit tumor invasion. Therefore, all these diverse responses suggest that activities of IFN are cell dependent but also dependent of the dose and the conditions of administration.

We provide evidence that the activation of caspases 1, 8, and 9 are involved in this apoptotic process, whereas IAP2 and BclxL did not. We showed that inhibition of both PI3k and NF-{kappa}B decreased IFN-{gamma}-induced apoptosis, whereas those of p38/MAPK and PKA increased the apoptotic effect of IFN-{gamma} (Table 2)Citation . These results suggest that IFN-{gamma}-induced cell death in RT112 cells could be regulated through PI3k and NF-{kappa}B, confirming their apoptotic role (23 , 33) , whereas P38/MAPK and PKA could have an antiapoptotic role. Thus, both p38/MAPK and PKA may be promising targets for bladder cancer therapy. The protein synthesis inhibitor CHX decreased IFN-{gamma}-induced apoptosis, suggesting that it was because of an inductive phenomenon. Furthermore, because cytokines act through binding to specific receptors, our results suggest that all of the bladder cancer cell lines express IFN-{gamma} receptor. Some of the transduction pathways are still functional (differentiation and cell growth), whereas those leading to cell death are lost or inactive (constitutive resistance to the cytotoxic effect of chemical agents).

We then investigated the effect of low doses of IFN-{gamma} (0.05–5 ng·ml-1) (4–400 IU·ml-1). These doses were compatible with those reported in the urine of bladder cancer patients after BCG therapy (34) . Because RT4 and RT112 cells responded to a low dose of exogeneous IFN-{gamma} (0.05 ng·ml-1), IFN-{gamma}-R are probably not activated constitutively through an autocrine loop. Pretreatment of the RT112 cells with a low dose of IFN-{gamma} for >24 h markedly inhibited their sensitivity to the cytotoxic effect of TNF-{alpha}, induced morphological changes, and increased cell motility and scattering. Inhibition of this process by CHX indicates that IFN-{gamma} activates synthesis of protein(s) that induces resistance to the cytotoxic effect of TNF-{alpha}, cell motility, and scattering of RT112 cells. Because HGF possess all these effects (24 , 35 , 36) , we investigated it and showed that HGF-like activities induced by IFN-{gamma} was inhibited by CHX. However, addition of specific neutralizing anti-HGF antibodies to IFN-{gamma}-treated cells only produced a partial phenotypic reversion, suggesting a continuous production and secretion of HGF. It’s also possible that IFN-{gamma} could induce a resistance process because of its action on the secretion of cytokines possessing this property (TNF-{alpha}, epidermal growth factor, fibroblast growth factor, transforming growth factor ß). Preliminary result shows that IFN-{gamma} did not induce transforming growth factor ß production (234 ± 10 versus 214 ± 10, respectively, in control and IFN-induced cultures; data not shown). However, our results also shown that IFN-{gamma} can induce low levels of TNF-{alpha} as recently published (21) , which can inhibit apoptotic process through several mechanisms, including manganese superoxide dismutase activity and NF-{kappa}B (20 , 22) . Thus, HGF is likely to be involved in our model reinforcing the notion that autocrine HGF could mediate the bladder cancer invasion and the resistance to the cytotoxic effect of chemical agents (24) . However, other cytokines induced by IFN-{gamma} (TNF-{alpha}) may also have occurred. The fact that RT4 cells treated with IFN-{gamma} did not secrete either HGF or TNF-{alpha}, neither acquire chemoresistance nor migrate, strengthen this hypothesis. In vitro treatment of tumor cells with IFN-{gamma} also leads to the modulation of their metastasizing ability (17 , 18) . In vivo, treatment with IFN-{gamma} reduced the incidence of pulmonary tumor metastases (37) , whereas in melanoma, disease-free survival was significantly worse in the group of IFN-{gamma}-treated patients (38) . The exact mechanisms that mediate these opposite effects are unknown, however, bladder tumor cells are able to secrete IFN-inducible protein 10 (an antiangiogenic chemokine) or MMP-2 or MMP-9 in response to BCG or IFN-{gamma} stimulation (31 , 32 , 39) . In our study, IFN-{gamma} increased the TNF receptor I antigen expression in both the RT4 and RT112 cells, whereas it induced a resistance to the cytotoxic effect of TNF-{alpha} in the RT112 cells and increased the sensitivity to TNF-{alpha} of the RT4 cells. This IFN-{gamma}-induced cell-specific response suggests that acquired resistance of the bladder cancer cell lines is not related to the cell death receptor expression, confirming that cytokine receptor expression did not correlate with the biological response (40) . The question remains of how IFN-{gamma} promotes either tumor invasion or tumor regression and apoptosis. Our results suggest that response is related to the IFN-{gamma} protein levels confirming clinical investigations showing that: (a) tumor-free patients produce higher BCG-induced IFN-{gamma} than tumor recurrence patients (6) ; and (b) T-helper 1 lymphocyte cytokines (IL-2 and IFN-{gamma}) profile was associated with the clinical response to BCG (41) . Moreover, IFN potentiates the therapeutic efficacy of paclitaxel (32) . These data support the hypothesis that the optimal injection of systemic IFN, associated with a BCG therapy that induce low IFN levels (10–200 IU·ml-1; Ref. 32 ), could induce an apoptotic process because of a sufficient dose (>400 IU·ml-1) as shown in our experiments.

In conclusion, our findings suggest that in addition to being an inflammatory cytokine, IFN-{gamma} may also induce apoptosis of bladder tumor cells through the activation of caspases 1, 8, and 9. Moreover, bladder cancer cell lines can respond to IFN-{gamma} by undergoing an epithelial to fibroblastic phenotype conversion together with induction of motility and resistance to the cytotoxic effect of TNF-{alpha} through Akt/PI3k and NF-{kappa}B pathways. This type of modulation can now be added to that observed in other experimental systems, showing that different doses of the same cytokine (CSF-1) can exert opposite effects (42) . Although our data suggest a great potential for IFN-{gamma} in therapy of solid tumors, extensive clinical trials have conclude that IFN-{gamma} are ineffective against most solid tumors (43) . Additional studies are required to better delineate the specific effects of PI3k in the expression of the gene targets able to induce either apoptotic or antiapoptotic process. The future development of anticancer immunotherapies is a challenge requiring the induction of an efficient immune response and avoidance of chemoresistance in bladder tumor cells.


   ACKNOWLEDGMENTS
 
We thank Linda Northrup for her help in English language editing, and Patrick Christophe for her expertise in microphotographic analysis.


   FOOTNOTES
 
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.

This work was supported by grants from the Direction de la Recherche Clinique (DRC) of the Centre Hospitalier Universitaire de Grenoble (France).

1 To whom requests for reprints should be addressed, at Laboratoire de Cytologie, Centre Hospitalier Universitaire de Grenoble, BP 217X, 38043 Grenoble Cedex 09, France. Phone: 33-4-76-76-54-89; Fax: 33-4-76-76-59-92; E-mail: PChampelovier{at}chu-grenoble.fr Back

2 The abbreviations used are: BCG, bacillus Calmette-Guerin; TNF, tumor necrosis factor; IL, interleukin; CSF-1, colony-stimulating factor 1; uPA-R, urokinase plasminogen activator receptor; HGF, hepatocyte growth factor; ICAM-1, intercellular adhesion molecule 1; PAI, plasminogen activator inhibitor; t-PA, tissue plasminogen activator; CS, culture supernatant; MnSOD, manganese superoxide dismutase; GPX, glutathion peroxidase; PDTC, pyrrolidinedithiocarbamate; PKA, protein kinase A; MAPK, mitogen-activated protein kinase; PI3k, phosphatidylinositol 3'-kinase; SB, SB203580; CHX, cycloheximide; PD, Parke-Davis compound PD098059; AcD, Actinomycin D; H7, 1(-5-isoquinolinylsulphonyl) 2 methylpiperazine; PI, propidium iodide; ICE, interleukin 1ß converting enzyme; IAP, inhibitor of apoptosis; MMP, matrix metalloproteinase; NF-{kappa}B, nuclear factor-{kappa}B. Back

Received 4/ 1/03; revised 6/ 2/03; accepted 6/ 3/03.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 B. Effects of Low...
 DISCUSSION
 REFERENCES
 

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