Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, D-50935 Cologne, Germany1
Author for correspondence: Sigrun Smola-Hess. Fax +49 221 478 3902. e-mail s.hess{at}uni-koeln.de
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Similar to multiple myeloma, IL-6 was postulated to be an autocrine growth factor for cervical carcinoma (Eustace et al., 1993 ). However, as shown previously, cervical carcinoma cells producing IL-6 in large quantities do not respond to it due to the loss of gp80 expression (Bauknecht et al., 1999
; Hess et al., 2000
). Detailed analysis revealed that other components of the signalling cascade are intact, as responsiveness to IL-6 could be completely restored by the addition of gp80 in a soluble form (Hess et al., 2000
). Cervical carcinoma cell lines like SW756, which produces nanogram amounts of IL-6, even responded to their own IL-6 in the presence of sgp80, leading to strong activation of STAT3 and production of the chemokine MCP-1 (monocyte chemoattractant protein-1). As MCP-1 may enhance an anti-tumour response by attracting mononuclear cells into the tumour tissue, loss of gp80 expression might be a helpful immune escape mechanism for the carcinoma cells (Hess et al., 2000
).
The effects of IL-6 signalling on human papillomavirus (HPV) regulation in cervical carcinoma cells are less well understood. Whereas IL-6 activated the HPV18 promoter in the hepatoma cell line HepG2, it was postulated that IL-6 might repress the HPV18 long control region (HPV18-LCR) in cervical carcinoma cells through the activation of c/EBP. However, this hypothesis could not be tested in the latter cells due to the fact that they have lost gp80 expression.
To further analyse this question we restored responsiveness to IL-6 by adding sgp80 to the IL-6-producing cervical carcinoma cell line SW756. Cells were kept in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 0·1 mg/ml streptomycin, 1 mM sodium pyruvate and 2 mM L-alanyl-L-glutamine (all from Gibco BRL). They were seeded at a density of 1·5x105 cells per well in 6-well plates, grown overnight and co-transfected with 1 µg of the vector p4321-luc expressing the luciferase gene under the control of the HPV18-LCR and 1 µg of CMV-galactosidase (CMV
-gal) plasmid as an internal control using the FuGene reagent (Boehringer Mannheim). The p4321-luc construct, kindly provided by Dr G. Steger, Cologne, Germany (Steger & Corbach, 1997
), is based on a HPV18-LCR construct, positions 6929 to 120, obtained from Dr Francoise Thierry, Paris, France. At 24 h post-transfection the cellular supernatants were replaced by medium with 100 ng/ml of IL-6, 500 ng/ml of sgp80 or a combination of both. As shown in Fig. 1
(lane 2), IL-6 did not alter HPV18-LCR activity. This was expected, as the cells do not express gp80. To our surprise, addition of sgp80 (Fig. 1
, lane 3), which restores IL-6 responsiveness, did not suppress, but instead activated the HPV18-LCR, similar to HepG2 cells (Bauknecht et al., 1999
). Apparently, the IL-6 produced by the SW756 cells themselves was sufficient for about 2·3-fold activation. Data shown are normalized to CMV
-gal stimulation. Due to the fact that this construct is also stimulated by IL-6, the presented data are weaker than non-normalized data. A similar result was obtained when IL-6 and sgp80 were combined for stimulation (Fig. 1
, lane 4).
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Earlier studies had revealed rapid and strong activation of STAT3 by sgp80 in SW756 cells (Hess et al., 2000 ). Computer analysis of the HPV18-LCR using the MatInspector program did not reveal a consensus STAT-binding site, which does not exclude the existence of an atypical yet functional STAT-binding site. Seven potential sites showing at least similarity with a STAT-binding site were investigated by EMSA and turned out not to bind STAT3 (data not shown). However, as STAT factors may undergo interactions with other transcription factors, e.g. STAT3 with c-jun (Schaefer et al., 1995
), and may regulate transcription without direct DNA binding (Niehof et al., 2000
), we were interested to investigate whether STAT factors are functionally involved in HPV-LCR activation. In order to block either STAT1 or STAT3 activation specifically, dominant-negative forms specific for the respective transcription factors were used. These STAT mutants (STAT1F and STAT3F) cloned into the vector pCAGGS-Neo (pCAGGS-Neo-HA-STAT1F and pCAGGS-Neo-HA-STAT3F) were kindly provided by Dr T. Hirano, Osaka, Japan (Hirano et al., 1997
; Nakajima et al., 1996
). In transient transfection analysis, dominant-negative STAT1 and STAT3 were tested for their ability to inhibit LCR activation by IL-6/sgp80. SW756 cells were seeded in 6 cm dishes. After co-transfection with 1 µg of the vector p4321-luc, 1 µg of CMV
-gal and 1 µg of pCAGGS-Neo-HA-STAT1F or pCAGGS-Neo-HA-STAT3F, respectively, SW756 cells were stimulated with 100 ng/ml IL-6 in the presence of 500 ng/ml sgp80 as described above. In contrast to STAT1F, STAT3F displayed a strong inhibitory effect (Fig. 3
). These results strongly indicated the involvement of the transcription factor STAT3 in IL-6/sgp80-mediated HPV18-LCR activation. Alternatively, overexpressed dominant-negative STAT3 might have titrated a different factor associated with STAT3.
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We are well aware of the fact that reporter construct analyses might not completely reflect the response of the integrated genes in carcinomas, as their regulation is undoubtedly much more complex and might also additionally depend on cellular factors acting at the viral integration sites. However, our study provides evidence that IL-6, produced by SW756 carcinoma cells, can clearly provide positive regulatory signals which induce the HPV18-LCR.
In conclusion, in cervical carcinoma cells a major role of shutting off the autocrine IL-6 stimulation might not be the repression of the viral oncogene promoter, but possibly an immunological role, e.g. to prevent chemokine production, which might help the malignant cells to escape the immune surveillance.
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Acknowledgments |
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Az FOR 265/2-1). S.S.-H. is supported by the Innovationsprogramm Forschung of the MSWWF Nordrhein-Westfalen.
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Received 4 April 2001;
accepted 8 June 2001.