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Experimental Therapeutics, Preclinical Pharmacology |
Induces Antiproliferative and Antitumoral Responses in Malignant Mesothelioma1
Equipe INSERM EMI.99.09 [F. G., Y. P., I. M., S. M., J. L. C., M-C. J.] and Unité INSERM U448 [C. B.], Faculté de Médecine Paris XII, 94010 Creteil Cedex, France; Service de Pneumologie, Centre Hospitalier Intercommunal, 94010 Créteil Cedex, France [I. M.]; and UMR INRA-AFSSA 1161, Ecole Nationale Vétérinaire d’Alfort, 94704 Maisons-Alfort, France [M. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene transfer on human mesothelioma (HM) cell proliferation in vitro and growth in nude mice.
Experimental Design: We constructed an E1E3-deleted replication-defective recombinant Ad carrying the human IFNgene (Ad-IFN) and tested its activity in vitro on HM cell lines established in our laboratory and in a nude mice model.
Results: In vitro, infection of HM cells with Ad-IFN led to a prolonged production of an active cytokine in the 10 HM cell lines tested and also led to an antiproliferative effect on the HM cells previously demonstrated as responsive to exogenous recombinant human IFN. In nude mice, s.c. inoculation of HM cells from one responsive HM cell line previously infected with Ad-IFN resulted in a delay in tumor development, and injection of Ad-IFN in preestablished tumors restrained tumor development.
Conclusions: These results indicate for the first time that HM cells are efficiently transduced by Ad-IFN and produce an active cytokine for several days. IFN produced by gene transfer is shown to have both an antiproliferative effect in vitro and an antitumoral effect in vivo in a nude mice model.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several authors have developed new strategies using antitumoral cytokines in MM (5
, 6)
, and interest in IFN for adjuvant or single therapy of various types of cancer, such as renal cell cancer, malignant melanoma, ovarian cancer, and non-small cell lung cancer, has been reported (7, 8, 9, 10)
. With regard to mesothelioma, the most impressive clinical results of cytokine therapy were obtained with intrapleural delivery of r-hu-IFN (5)
. However, r-hu-IFN efficiency was demonstrated mostly in early stages of MM and was limited by the short half-life of the recombinant cytokine (6)
. Thus, further investigations for developing methods allowing continual treatment of MM are required.
Among potential candidates, gene therapy is likely to be useful in MM, and a surgical debulking procedure to remove gross disease, followed by gene therapy to remove residual disease, would be technically feasible (5)
. Thus far, several recombinant Ads have been constructed carrying various genes of interest, such as interleukin-12 (11)
, P16/INK4a (12)
, P14(ARF) (13)
, BAK (14)
, or P53 (15)
. Moreover, a suicide gene strategy using an Ad-TK has given encouraging results, showing minimal side effects (16)
. Viral constructs carrying the IFN gene showed efficiency in animal models of diverse tumors (17
, 18)
and in patients with metastatic melanoma (19)
, but to date, gene transfer of IFN has never been applied to MM.
Based on these data and on previous reports from our laboratory showing an IFN-induced inhibition of cell proliferation in HM cells in vitro (20
, 21)
, we developed a recombinant Ad carrying the IFN gene (Ad-IFN). The mechanisms whereby IFNs exert their growth-inhibitory effect are not well understood. Earlier findings suggested that IFN may act by controlling metabolic pathways involving activation of indoleamine-2,3-dioxygenase and NO synthase (22)
. However, these pathways did not appear to be involved in r-hu-IFN-induced HM cell cytostasis (23)
. Recently, several pathways have been suggested, involving IRF-1-regulated genes, antagonism with the action of growth factors, regulation of proteins that regulate cell cycle progression (up-regulation of cyclin-dependent kinase inhibitors and down-regulation of regulatory cyclin subunits; Ref. 24
), and up-regulation of proapoptotic genes (25)
. In HM cell lines, IFN produces an arrest in G1 and G2-M phases of the cell cycle, and the down-regulation of cyclin A has been suggested to play a role (26)
. No apoptosis was detected. Moreover, from the study of resistant cell lines, it seems that IRF-1 activation is important to account for the antiproliferative action of IFN in HM cells (21)
. Thus, IFN could possibly produce an in vivo antitumor response through a direct antitumor effect and/or through an indirect mechanism involving immunomodulation.
The present work had two objectives: first, to determine whether Ad-mediated IFN transfection resulted in an antiproliferative effect on HM cell lines; and second, to evaluate the effect of transfection on the growth of these human cells in a nude mice model. Our results demonstrate a strong infectability and transduction of mesothelioma cells and, more importantly, a direct antiproliferative effect of the transduction product both in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene. was constructed as described previously (27)
. The human IFN cDNA PTG13 was inserted into the SmaI-NheI sites of a pCI plasmid (Promega, Charbonnières, France) under the control of the CMV immediate early promoter/enhancer and containing a chimeric intron and a downstream polyadenylation signal from SV40. Ad-TK and Ad-ßGal were prepared in parallel. The titer of recombinant virus stock was determined by plaque assay on 293 cells and expressed as the TCID50 per ml. In some experiments, Ad encoding GFP (Ad-CMV-GFP) was also used.
Cell Lines.
HM cell lines were cultured according to standard methods used in the laboratory, as described previously (21)
, in RPMI 1640 supplemented with 25 mM HEPES, 8% fetal bovine serum, 80 µg/ml streptomycin, and 80 IU/ml penicillin (all from Life Technologies, Inc., Cergy-Pontoise, France). Ten HM cell lines were selected for the present study, according to previous results on signal transduction and growth response to r-hu-IFN (20
, 21)
: eight cell lines (BT, DV, HB, MR, QR, BL, RV, and BN) were growth-inhibited after treatment with r-hu-IFN (Imukin; Boehringer Ingelheim, Paris, France), and two cell lines (CR and FR) did not respond to r-hu-IFN. Henceforth, they will be referred to as "responsive" and "unresponsive," respectively.
Cell line 293 expressing adenoviral genes was obtained from the American Type Culture Collection. WISH cells were provided by J. Wietzerbin. Both cell lines were routinely cultured in DMEM (Life Technologies, Inc.), supplemented as described for RPMI 1640.
Cell Infection and Collection of Conditioned Medium.
HM cells were dispensed in 24-well tissue culture plates (Costar; Dutscher, Brumath, France; 140,000 cells/ml/well). The desired amount of recombinant Ad was added to the cultures 24 h after plating, on a per cell basis. The plates were incubated at 37°C in 95% air, 5% CO2 for 1.5 h. Thereafter, the culture medium was removed and replaced with fresh medium, and plates were incubated for the indicated time. Then the conditioned medium was removed, centrifuged at 300 x g to get rid of cell debris, and stored at -20°C until further use.
IFN Production by Ad-IFN-infected HM Cell Lines.
IFN concentration was measured in duplicate with an ELISA (Quantikine TM; R&D Systems, Abingdon, United Kingdom) in the conditioned media from Ad-IFN-treated HM cells, according to the manufacturer’s recommendations. The activity of IFN produced by gene transfer was assayed by inhibition of the cytopathic effect of vesicular stomatitis virus on human WISH cells (28)
.
In Vitro Investigation of the Antiproliferative Effect of IFN Produced by Gene Transfer.
The effect of Ad-IFN infection on HM cell growth was determined in the 24-well plates used to collect conditioned medium for ELISA measurement of IFN concentration, as described above. The effect of conditioned medium from Ad-IFN-treated cells on HM cell growth was evaluated according to the following procedures: HM cells were dispensed into 24-well tissue culture plates at a concentration of 1.5–4 x 104 cells/ml/well, depending on the cell line. After incubation for 24 h at 37°C in 5% CO2, the cells were either exposed to conditioned medium from cells previously infected with 100 TCID50 Ad-IFN/cell or cocultured with infected HM cells from the same or a different HM cell line. In coculture assays, HM cells were first plated on a Transwell membrane (Dutscher, Brumath, France) in a 24-well multiwell plate and then infected with 50 TCID50 Ad-IFN/cell and incubated under standard conditions. After 24 h, the membranes were washed with complete culture medium and transferred into 24-well tissue culture plates containing the untreated growing HM cells.
Cell proliferation was investigated using a MTT assay (20) . The absorbance was measured at 540 nm using an automated microplate reader (EL 800; BIO-TEK Instruments, Fischer Scientific).
Effect of Ad-IFN Infection on the Tumorigenic Potency of HM Cell Lines.
Female nude mice (Swiss nu/nu; 7 weeks old) were obtained from Iffa Credo (L’Arbresle, France) and housed according to European Union Guidelines (29)
. After 1 week of adaptation, groups of six or seven mice were inoculated s.c. in the scapular region with 3 x 106 HM cells, which were either untreated or previously infected with 50 TCID50 Ad-IFN/cell. Two HM cell lines were tested: one responsive cell line (BT) and one nonresponsive cell line (FR). Tumor volume (V) was measured twice a week, according to the formula: V = L x W2/2 (L, length; W, width). After sacrifice, tumors were either fixed in 10% formalin or cultured to control their responsiveness to r-hu-IFN using the MTT assay.
Effect of Ad-IFN Treatment on Growth of Pre-established Tumors.
Two groups of 24 female nude mice (Swiss nu/nu; 7 weeks old) were inoculated s.c. in the scapular region with 3 x 106 cells from BT and FR cell lines. Six mice inoculated with PBS served as control. Tumor nodules were visible after less than 1 week. Three weeks after inoculation, all tumors were >40 mm3. Each treatment was randomly attributed to six mice in each group: 2 x 108 TCID50 of recombinant Ad (Ad-IFN, Ad-TK, or Ad-ßGal) or PBS alone was injected intratumorally in 50 µl of PBS. Tumor volumes were measured twice a week.
Statistical Analyses.
Results were evaluated using Dunnett’s, log-rank tests and linear regression with Graph Pad Prism Software V2.0 for Macintosh. The difference was considered significant when P 
0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production by Ad-IFN-infected HM Cell Lines. cumulative production over the course of 6 days. All cell lines produced IFN after infection (Fig. 1A)
. All cell lines previously found to be responsive to r-hu-IFN produced amounts of IFN greater than their IC30 (concentration of r-hu-IFN reducing growth by 30%; Ref. 20
).5
No IFN was found in the culture medium from uninfected cells.
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at day 7, but the production was lower than that seen 3 days after infection. At day 14, IFN was still detected in the conditioned medium from four HM cell lines (DV, RV, BN, and FR). Furthermore, the FR cell line still produced IFN 28 days after infection (data not shown).
Four cell lines, BT, BL, HB and MR, were treated with two different doses of Ad-IFN, and subsequent production of IFN was compared. Increasing the Ad-IFN concentration from 50 to 100 TCID50/cell resulted in more than a doubling of the IFN production (Fig. 1B)
.
Activity of IFN Produced by Gene Transfer.
To compare the effect of IFN produced by gene transfer with that of r-hu-IFN produced by Escherichia coli, the specific activity of the IFN produced by gene transfer was determined in the conditioned medium from three HM cell lines (BT, BL, and FR) collected 24 h after infection with 100 TCID50 Ad-IFN/cell. The r-hu-IFN (20 IU/ng) from Boehringer Ingelheim was used as standard. The activity of IFN produced by gene transfer was found to be 11, 24, and 61 IU/ng in the HM cell lines FR, BL, and BT, respectively.
Antiproliferative Action of IFN Produced by Gene Transfer.
Mesothelioma cell growth was significantly reduced in the cultures treated with Ad-IFN (Fig. 2)
, except in two HM cell lines (CR and FR) previously demonstrated to be unresponsive to r-hu-IFN (20
, 21)
. No antiproliferative effect was observed after infection with Ad-ßGal (data not shown). Two cell lines (BT and BL) were also cultured in conditioned medium from the same or a different HM cell line or cocultured with cells of the same or a different HM cell line previously infected with 100 TCID50 Ad-IFN/cell. Cell proliferation was inhibited in comparison with the control cultures (Fig. 3)
.
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Concentration on the Proliferation of HM Cell Lines.
shows that the cell growth was significantly reduced in the responsive cell line in a dose-dependent manner, but not in FR.
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-treated HM Cell Lines.
shows the time course of the percentage of tumor-free mice after treatment with BT and FR cells. All animals inoculated with uninfected cells from the BT cell line exhibited nodules 12 days after inoculation, whereas mice inoculated with Ad-IFN-infected BT cells did not develop nodules earlier than 58 days after inoculation. Time between inoculation and tumor appearance was found to be statistically different between treated and untreated cells (log-rank test). In contrast, both infected and noninfected FR cells produced nodules without significant difference in the time of tumor appearance. To determine whether the delayed growth of the Ad-IFN-treated BT cells could be due to a phenotypic reversion (resistance to IFN), we verified that the in vitro growth of cells cultured from the tumors was still inhibited by r-hu-IFN (data not shown).
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Treatment on Growth of Preestablished Tumors.
shows the time-dependent evolution of tumor volumes in mice inoculated with the BT cell line: tumors treated with Ad-IFN grew significantly slower than those treated with control Ads (linear regression, P < 0.01). Tumors treated with PBS grew significantly faster than all of the others (P = 0.011). For mice inoculated with FR cells, tumor development was also significantly reduced after all treatments, when compared with PBS-treated tumors (linear regression, P < 0.001; Fig. 6B
). However, no significant difference among the three treatments (i.e., Ad-ßGal, Ad-TK, or Ad-IFN) was observed (P > 0.08).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production and inhibition of cell proliferation and tumor growth in different HM cell lines after infection with an E1E3-deleted recombinant Ad encoding human IFN. We observed that HM cell lines were all able to produce levels of IFN that were previously shown to have an antiproliferative action in responsive cells (20)
. However, IFN production differed between HM cell lines (4–2306 ng produced over a 6-day period, for instance). The differences in IFN production could not be attributed to different infectabilities because the percentage of cells transduced after exposure to 100 TCID50/cell, on the basis of the results obtained with Ad-GFP, was similar between the cell lines (data not shown). However, the degree of GFP expression did not correlate with the degree of IFN production. Therefore, differences in the mechanisms leading to gene expression in the different cell lines could account for these results.
In vitro, all of the HM cell lines tested were growth-inhibited after infection with Ad-IFN, with the exception of CR and FR, which were previously found to be unresponsive to r-hu-IFN because they have an impaired Janus-activated kinase/signal transducers and activators of transcription signal transduction pathway, i.e., FR was shown to lack Janus-activated kinase 2 expression, whereas a poor activation of IRF-1 was demonstrated in CR (21)
. Interestingly, all sensitive cell lines produced amounts of IFN higher than their IC30 and were, accordingly, more than 30% growth-inhibited. Cell growth inhibition observed in vitro likely results from a specific effect of IFN, as suggested by different findings. Firstly, no growth inhibition was observed after treatment with Ad-ßGal. Secondly, growth inhibition in the presence of conditioned medium from cells infected with Ad-IFN and coculture assays demonstrated that the medium from transfected cells exerted a cytostatic effect that was not observed with conditioned medium from uninfected cells.
Intracavitary therapy has become accepted for treatment of MM because it permits a greater concentration of drug within the cavity compared with systemic administration. However, the half-life of IFN appears limited, likely because of its degradation (30)
. Thus, local production of IFN at the tumor site deserves to be investigated. From the time course of IFN production, we concluded that transfection of HM cells permits a longer exposure to IFN because all HM cell lines still produced a detectable amount of the transgene 7 days after infection. Furthermore, IFN was still detected in four HM cell lines 14 days after infection and was detected in FR even 28 days after infection. However, a progressive time-dependent decrease in IFN production was observed. A time-dependent decrease in the amount of IFN transcript was also found, except in the FR cell line, as demonstrated by quantitative real-time PCR analysis (data not shown). This could be due to a slowdown of the cell machinery induced by IFN and perhaps to the lack of fresh media because the medium was not changed through a 6-day period, or it could be explained by the CMV promoter being down-regulated by IFN (31)
. This decrease may also be related to a dilution effect of the vector because the cells did not stop growing immediately after infection but stopped growing at least 2 days later (data not shown). Such a delay was also observed with direct addition of r-hu-IFN (20
, 21)
. A time-dependent decrease in IFN expression was also found in fibroblasts infected with Ad-IFN (32)
.
In immunocompetent animals, Ad-IFN can stimulate the host immune response against the tumor cells (17)
and then have both a direct antiproliferative effect and an indirect immune effect toward the tumor cells. Alternatively, Ad-IFN has been demonstrated to repress growth of established brain tumors by antiangiogenesis (18)
. Thus, given the pleiotropic effects of Ad-IFN, some response could be also obtained in vivo with cells unresponsive to the antiproliferative effect of IFN.
Activity of IFN produced by gene transfer ranged from 11 to 61 IU/ng in three HM cell lines studied. This value was of the same order as that of r-hu-IFN (20 IU/ng) used in clinical trials (6)
. The differences in IFN activity between cell lines could be related to different proteolytic activities in the culture medium between the cell lines, possibly leading to differences in the activation or inactivation of the cytokine (30)
.
Because immunocompetent animals cannot be used to investigate the effect of drugs on human cells, the nude mice model appears useful for assessing the effect of drugs (33)
. Injection of in vitro-transfected BT cells resulted in a significant delay in tumor development when compared with untreated BT cells, in agreement with the antiproliferative action of IFN on this cell line. In contrast, no change in tumor incidence and delay of tumor formation was observed in the cell line FR that did not display an antiproliferative response to IFN. This result suggests that the antiproliferative action demonstrated in vitro also occurred in vivo and produced a delay in tumor growth. We found that the lack of duration in the inhibition of tumor development in mice inoculated with Ad-IFN-infected BT cells was not caused by a selection of resistant cells. This effect was more likely due to a dilution of the transgene along with cell division and/or to a down-regulation of the gene promoter, as discussed above. Alternatively, the persistence of the expression seems to be dependent on the vector backbone and host background in a tissue-specific manner (34)
.
When Ad-IFN was inoculated in preestablished tumors, a significant decrease in tumor growth was observed with both cell lines tested when compared with PBS. However, with the cell line unresponsive to IFN, the inhibitory effect observed with Ad-IFN was not significantly different from that of control Ads. In contrast, in the responsive cell line, Ad-IFN was significantly more efficient in reducing tumor growth than control Ads. An inhibitory effect of nonrelevant Ads has been described elsewhere in immunocompetent animals and could be explained in our system by an activation of the residual immune system of nude mice, including natural killer cells (35)
and macrophages.
Additional studies are still needed to understand the mechanisms involved in the antiproliferative and antitumoral effects of IFN produced by gene transfer and to determine the best conditions for a possible treatment of human MM by IFN gene therapy. Moreover, experimental trials are necessary before testing the promising new therapy on human patients to be as efficient and safe as possible (36)
.
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ACKNOWLEDGMENTS |
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cDNA. Ad-CMV-GFP was kindly provided by B. Klonjkowski (Ecole Nationale Vétérinaire d’Alfort). We are grateful to S. Legouvello (Laboratoire d’Immunologie Biologique, CHU Henri Mondor) for allowing us to use the light cycler system and to C. Batisse (EMI 99.09) for technical help. |
FOOTNOTES |
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1 The authors were supported by funds from INSERM, Ministère de la Recherche and grants from the Association pour la Recherche sur le Cancer (7589) and from the Ligue Nationale contre le Cancer, Comité du Val d’Oise. F. G. is a fellow of the Ligue Nationale contre le Cancer, Comité du Val de Marne and of the Association pour la Recherche sur le Cancer. 
2 Present address: Unité INSERM U402, Faculté Saint-Antoine, 75012 Paris, France.
3 To whom requests for reprints should be addressed, at INSERM EMI 99.09, Faculté de Médecine, Université Paris XII, 8 rue du Général Sarrail, 94010 Creteil Cedex, France. Phone: 33-1-49-81-36-56; Fax: 33-1-49-81-35-33; E-mail: jaurand{at}im3.inserm.fr
4 The abbreviations used are: MM, malignant mesothelioma; Ad, adenovirus; HM, human mesothelioma; r-hu-IFN, recombinant human IFN; TK, thymidine kinase; IRF, interferon regulatory factor; CMV, cytomegalovirus; TCID50, 50% tissue culture infective dose; GFP, green fluorescence protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ßGal, ß-galactosidase.
5 L. Zeng and M. C. Jaurand, unpublished data.
Received 12/ 5/01; revised 6/13/02; accepted 6/17/02.
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) and 100 TCID50 (
) Ad-IFN
). B, cells were exposed to the conditioned medium from the same cell line (
). The results are expressed as the percentage of absorbance value in the treated culture, in comparison with untreated culture. Difference was assessed by Dunnett’s test (treated versus untreated): *, statistically significant; ns, not significant.
). Cell growth was determined with the MTT assay. Results are expressed as the percentage of absorbance value obtained in the MTT assay in comparison with untreated cells. Difference was assessed by Dunnett’s test (treated versus untreated): *, statistically significant; ns, not significant.
) either infected (squares) or not infected (circles) with 50 TCID50 Ad-IFN
), Ad-ßGal (•), Ad-TK (