JOURNAL OF VIROLOGY, Jan. 2002, p. 280–291 Vol. 76, No.

1
0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.1.280–291.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Disturbance of Tumor Necrosis Factor Alpha-Mediated Beta

Interferon Signaling in Cervical Carcinoma Cells
Anastasia Bachmann, Brigitte Hanke, Rainer Zawatzky, Ubaldo Soto, Jan van Riggelen,
Harald zur Hausen, and Frank Rösl*
Forschungsschwerpunkt Angewandte Tumorvirologie, Abteilung Tumorvirus-Immunologie, Deutsches
Krebsforschungszentrum, Heidelberg, Federal Republic of Germany
Received 3 July 2001/Accepted 28 September 2001

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In the present study we show that malignant human papillomavirus (HPV)-positive cells lost their ability to
synthesize endogenous beta interferon (IFN-␤) upon tumor necrosis factor alpha (TNF-␣) treatment. IFN-␤
transcription, however, was reinducible in nonmalignant HPV-positive cells, which was confirmed in functional
protection assays against encephalomyocarditis virus or vesicular stomatitis virus infections. Addition of
neutralizing antibodies against IFN-␤ blocked the antiviral effect, excluding the possibility that other IFN types
were involved. Conversely, both malignant and immortalized cells could be protected against viral cytolysis
when either IFN-␤, IFN-␣, or IFN-␥ was added exogenously. This indicates that only the cross talk between
TNF-␣ and the IFN-␤ pathways, and not IFN-␣/␤ and IFN-␥ signaling in general, is perturbed in cervical
carcinoma cells. Notably, full virus protection was restricted exclusively to nonmalignant cells, indicating that
the antiviral effect correlates with the growth-inhibitory and virus-suppressive properties of TNF-␣. The
IFN-regulatory factors IRF-1 and p48 (ISGF3␥) emerged as key regulatory molecules in the differential IFN-␤
response, since their transcription was either absent or only inefficiently enhanced in tumorigenic cells upon
treatment with TNF-␣. Inducibility of both genes, however, became reestablished in cervical carcinoma cells,
which were complemented to nontumorigenicity after somatic cell hybridization. Complementation was par-
alleled by the entire reconstitution of cytokine-mediated IFN-␤ expression and the ability of TNF-␣ to exert an
antiviral state. In contrast, under conditions where tumor suppression was not accomplished upon somatic cell
hybridization, neither expression of IRF-1, p48, and IFN-␤ nor antiviral activity could be restored.

Human papillomavirus (HPV)-induced carcinogenesis is a
multistep process which is initiated by viral infection (68). At
what time and to what extent tumor formation takes place,
however, are dependent mainly on the immunological status of
the patient. As deduced from epidemiological data, immuno-
suppressed individuals or persons with impaired immunocom-
petence have a significantly higher risk for developing cervical
cancer than corresponding age-matched controls (53). Hence,
tumor appearance can be regarded in part as the result of an
immunological escape process during which either certain in-
ter- and intracellular surveillance mechanisms are functionally
abolished or HPV-positive cells are no longer susceptible to
immunological control (69). It is therefore reasonable to as-
sume that only a physiologically intact communication pathway
between inflammatory and HPV-containing cells guarantees a
proper antiviral response (52).
Indeed, when fresh biopsies from patients were evaluated by
immunohistochemistry, cervical cancer sections were found to
be significantly depleted of infiltrating macrophages, T lym-
phocytes, and dendritic cells compared with premalignant tis-
sue specimens (22, 60, 63, 64). The recruitment of immuno-
logical effector cells in turn is mediated by chemokines
(chemotactic cytokines) such as monocyte-chemotactic pro-
tein-1 (MCP-1) (39, 45, 47). Consistent with the notion that
dysregulation in intercellular communication may favor the
* Corresponding author. Mailing address: Angewandte Tumorvi-
rologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld
242, 69120 Heidelberg, Federal Republic of Germany. Phone: 49-
6221-42-4900. Fax: 49-6221-42-4902. E-mail: F.Roesl@DKFZ.de.
outcome of neoplasias, all cervical carcinoma lines tested up to
now are devoid of significant inducible MCP-1 expression.
Cytokine inducibility, however, can be completely restored in
somatic cell hybrids with normal cells (51), which were no
longer tumorigenic when heterotransplantated into immuno-
compromised animals (61). Tumorigenic segregants derived
from the same hybrids again lost MCP-1 expression (28, 51),
strongly suggesting that elimination of chemokine expression
may provide a selective advantage for tumor formation (52). It
should be stressed that this correlation is not restricted to
tissue culture conditions, because in situ hybridization studies
in combination with immunohistochemistry techniques con-
firmed that MCP-1 expression and infiltrating cells of the
monocyte/macrophage lineage were detectable only in prema-
lignant precursor cells and were absent in high-grade lesions of
cancer patients (29, 46).
Recruitment and activation of macrophages can be consid-
ered the first line of defense against generalized virus infec-
tions and viral spread (for reviews, see references 17 and 19).
For example, spontaneous regression of benign warts is accom-
panied by a strong infiltration of mononuclear cells (41), where
papilloma shrinkage directly correlates with high tumor necro-
sis factor alpha (TNF-␣) expression in surrounding macro-
phages (20). Hence, TNF-␣ not only may represent a key
regulatory cytokine in regression of benign tumors (20) but
also could play a pivotal role in the immunological control of
dysplastic cervical lesions infected with high-risk HPV types
such as HPV type 16 (HPV16) or HPV18. Although not yet
directly demonstrated in patients, it is conceivable that mac-
rophage-specific TNF-␣ synthesis can trigger paracrine MCP-1

280
VOL. 76, 2002 ABSENCE OF IFN-␤ EXPRESSION IN CERVICAL CARCINOMA CELLS
gene expression in immortalized cells (and vice versa) which in
turn augments mononuclear cell infiltration as well as secretion
of larger amounts of growth-inhibitory cytokines. In support of
this assumption was the finding that cocultivation with acti-
vated macrophages from normal human volunteers can both
suppress viral transcription and induce MCP-1 gene expres-
sion, but only in nonmalignant HPV-positive cells (51). Con-
versely, cervical carcinoma cells were completely refractory,
despite functional TNF-␣ signaling, as monitored by rapid
proteolysis of I␬B␣ upon cytokine application (13). The block-
age of MCP-1 synthesis and the concomitant resistance to
TNF-␣ may therefore perturb the intercellular cross talk and
favor the accumulation of malignant cells (52).
Another interesting property of TNF-␣ is its ability to confer
an antiviral state through induction of the beta interferon
(IFN-␤) gene (23, 38). IFN-␣/␤ production is the most rapid
host immune response against a variety of viral infections (9).
Moreover, it also became evident that the elimination of virus-
positive cells is not entirely exerted through major histocom-
patibility complex class I-restricted CD8⫹ cytotoxic T lympho-
cytes but also relies on appropriate temporal and local
expression of cytokines such as TNF-␣ and downstream IFN
signaling (19, 35).
Considering this scenario in the context of the transforming
potential of high-risk HPVs, there are a quite substantial num-
ber of studies which show that the IFN signal transduction
pathway is a target for viral oncoproteins E6 and E7. For
example, HPV18 E6 can interfere with the IFN-␣ response in
human fibrosarcoma HT1080 cells by impairing Jak-STAT1/2
tyrosine phosphorylation after binding of the ligand to the
corresponding receptor (32). Furthermore, E7 expression in
spontaneously immortalized human keratinocytes (HaCaT
cells) can inhibit p48 (ISGF3␥) protein translocation into the
nucleus (3). p48 (ISGF3␥) is part of IFN-stimulated gene fac-
tor 3 (ISGF3), a trimeric trans-activating complex formed be-
tween p48, STAT1, and STAT2 which induces transcription of
IFN-regulated genes via binding to cognate IFN-stimulated
response elements (5). However, all of these properties of the
viral oncogenes do not provide a reliable explanation of why
IFN still has a curative and growth-inhibitory effect on prema-
lignant HPV-positive cells (6, 14, 25), despite the fact that E6
and E7 are found to be expressed in those lesions.
To gain insight into IFN signaling and the antiviral activity of
TNF-␣, we used cervical carcinoma cells and malignant and
nonmalignant somatic cell hybrids derived therefrom as a
model system (61). For example, HPV18-positive HeLa cells
can be complemented to nontumorigenic phenotype growth
after somatic cell hybridization with normal human fibroblasts
or keratinocytes. Occasionally, however, rare tumorigenic seg-
regants of the same hybrids arise, which is accompanied by the
nonrandom loss of chromosomes, in particular of chromosome
11 (for a review, see reference 52). In the case of HPV18-
positive cervical carcinoma cells, chromosome 11 indeed seems
to play a key role in malignant transformation, since the rein-
troduction of the corresponding normal allele via microcell
transfer is sufficient to completely suppress tumor formation of
either the parental cells or the tumorigenic segregants (52a).
Here we demonstrate that, similar to the case for the MCP-1
gene (28, 51), IFN-␤ expression could be induced by TNF-␣
only in nonmalignant HPV-positive cells. The functionality and
281
specificity of endogenous IFN-␤ production were assessed in
antiviral protection assays using encephalomyocarditis virus
(EMCV) or vesicular stomatitis virus (VSV) as infectious
agents. In contrast, all malignant cells remained protected
against viral cytolysis when IFN-␤ was exogenously supple-
mented. This indicates that the disturbance of the TNF-␣-
mediated IFN-␤ expression and the loss of an immediate an-
tiviral response are additional central events in the multistep
progression to cervical cancer, being more the consequence of
the in vivo phenotype of the respective host cell rather than a
direct effect of E6 and E7 oncoprotein expression.
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MATERIALS AND METHODS
Cell lines, hybrid formation, and cytokine treatment. The cervical carcinoma
cell lines HeLa, SW756, CaSki, and SiHa; the nontumorigenic somatic cell
hybrids made between HeLa cells and normal human fibroblasts (444 cells) and
their tumorigenic segregants (CGL3 cells) (61); HeLa ⫻ CaSki and HeLa ⫻
SW756 hybrids (15, 59); SiHa ⫻ HeLa hybrids (see below); malignant HPK Ia
cells (10); and the human lung carcinoma cell line A549 (66) were maintained in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum
and 1% penicillin-streptomycin. Modified SiHa cells (so-called universal fuser)
(50) harboring dominant (G418 resistance) and recessive (hypoxanthine-phos-
phoribosyltransferase-negative) phenotypes were grown in the same medium
containing 1 mg of G418 (Gibco BRL) per ml and 10⫺4 M 6-thioguanine
(Sigma). Somatic cell hybridization between SiHa and HeLa cells was done
exactly as described previously (50). For cytokine treatment, the cells were
incubated with TNF-␣ (Strathman Biotech GmbH, Hannover, Germany), IFN-␣
(Alphaferon3 [natural human IFN-␣; kindly provided by Rentschler, Laupheim,
Germany]), IFN-␤ (IFN-␤1; [natural human IFN-␤; Rentschler), and IFN-␥
(IF-RC [Thomae, Biberach, Germany]) for different periods of time as described
in the figure legends.
RNA analysis. RNA was isolated with the RNeasy kit according to the instruc-
tions of the manufacturer (Qiagen, Hilden, Germany). Approximately 5 ␮g of
RNA was separated on 1% agarose gels in the presence of ethidium bromide
under nondenaturing conditions (26) and transferred to GeneScreen Plus mem-
branes (DuPont, NEN). Specific probes for the hybridization were labeled with
[32P]dCTP by random priming (12). The filters were washed in 2⫻ SSC (1⫻ SSC
is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% sodium dodecyl sulfate (SDS)
at 68°C and exposed to Kodak films as indicated in the figure legends.
RT-PCR. DNA digestion of total RNA was carried out with 2 U of RQ1-
DNase (specific activity, 1 U/␮l) (Promega, Mannheim, Germany) in 40 mM
Tris-HCl (pH 8.0)–10 mM MgSO4–1 mM CaCl2 in the presence of 20 U of
RNase inhibitor (specific activity, 40,000 U/ml) (Hybaid, Heidelberg, Germany)
in a total volume of 100 ␮l at 37°C for 10 min. DNase was inactivated by addition
of 2 ␮l of 0.5 M EDTA (pH 8.0), followed by acidic phenol-chloroform extraction
(8). RNA was diluted in RNase-free water. One microgram of DNase-treated
RNA was mixed with 0.5 ␮g of oligo(dT) primer (Promega) added in a total
volume of 12 ␮l, heated at 70°C for 10 min, and chilled on ice. The mixture was
supplemented with 5⫻ reverse transcription (RT) buffer (250 mM Tris-HCl [pH
8.3], 375 mM KCl, 15 mM MgCl2), 10 mM dithiothreitol, 500 ␮M deoxynucleo-
side triphosphate mix (Roche Diagnostic, Mannheim, Germany), and 20 U of
RNase inhibitor and incubated at 42°C for 2 min in a total volume of 19 ␮l. After
the annealing, 200 U of reverse transcriptase SuperScript II (Gibco BRL) (spe-
cific activity, 200 U/␮l) was added and the reaction mixture was incubated for 1 h
at 42°C. The RT products were heated to 72°C for 10 min and chilled on ice.
PCRs were performed in a solution containing 10 mM Tris-HCl (pH 8.3), 200
␮M deoxynucleoside triphosphate mix (Roche Diagnostic), 40 pmol of upstream
and downstream primers, 5 U of Taq polymerase (Sigma) (specific activity, 5
U/␮l), and 2 ␮l of reverse-transcribed product. The amplification was performed
in an MJ Research PTC-200 thermal cycler in a total volume of 50 ␮l. For IFN-␤
detection, the primers 5⬘-GATTCATCTAGCACTGGCTTG-3⬘ and 5⬘-CTTCA
GGTAAATGCAGAATCC-3⬘ (23) were used. The PCR was performed for 40
cycles consisting of 1 min at 94°C, 45 s at 55°C, and 30 s at 72°C. During the last
cycle, the extension time was increased to 10 min, and the reaction mixture was
rapidly cooled to 4°C. IFN-regulatory factor 3 (IRF-3) expression was monitored
using the upstream primer 5⬘-GGTTGCGTTTAGCAGAGGAC-3⬘ and the
downstream primer 5⬘-AGGAGATGGTCTGCTGGAAG-3⬘. Amplification (35
cycles) was done at 94°C for 30 s, 57°C for 45 s, and 72°C for 30 s. In the last cycle,
the extension time was increased (to 10 min) and then the mixture was rapidly
cooled to 4°C. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used
282 BACHMANN ET AL. J. VIROL.

as an internal control with primers 5⬘-TGGATATTGTTGCCATCAATGAC

C-3⬘ and 5⬘-GATGGCATGGACTGTGGTCATG-3⬘ (18). The amplification
was performed for 35 cycles of 1 min at 94°C, 45 s at 65°C, and 30 s 72°C, with
a prolonged extension time of 10 min in the last cycle before cooling to 4°C. The
PCR products were analyzed in 1 to 2% agarose gels.
Antiviral titration assays. Cells were infected with either EMCV or VSV using
multiplicities of infections (MOIs) of between 0.1 and 0.2 (37, 66). The cells,
seeded in 96-well plates (104 cells/well) were pretreated with cytokines as de-
scribed in the figure legends. After 24 h, the medium was discarded, fresh
medium (37°C) with virus was added, and the plates were incubated at 37°C until
the viral lysis in the controls was complete (16 to 20 h). All cells were susceptible
to EMCV, except CaSki ⫻ HeLa hybrids. For those cells, antiviral activity was
monitored after infection with VSV. To quantify the cytopathic effect (CPE), the
cells were fixed with 4% glutardialdehyde and stained with 1% crystal violet. The

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dye was solubilized in 33% acetic acid, and the optical density of the eluate was
measured at 570 nm in a Labsystem Multiscan MS enzyme-linked immunosor-
bent assay reader. To quantify the amount of endogenously synthesized IFN-␤ in
444 cells after treatment with TNF-␣, the supernatants were transferred on A549
indicator cells (66) (3 ⫻ 104/well). On the next day, the cells were infected with
EMCV. After 26 to 30 h, medium was removed and the cytopathogenic effect was
quantified as described above. The amounts of IFN-␤ are expressed as interna-
tional reference units per milliliter, using exogenously added National Institutes
of Health human IFN-␤ as a reference.
Neutralization assay. Cells (104) were seeded in 96-well plates. Prior to the
addition of TNF-␣, as indicated in the figure legends, the cells were incubated for
1 h at 37°C with antibodies neutralizing 8 U of IFN-␤ (clone 39C; Biotrend, Köln,
Germany) or 10 U of IFN-␣ (Hoffmann La Roche Inc., Basel, Switzerland).
After 24 h, medium was discarded and the cells were infected with EMCV and
incubated at 37°C until viral controls showed complete lysis. Cells were fixed with
4% glutardialdehyde and stained with 1% crystal violet. The eluate was quanti-
fied as described above.
EMSAs. For gel retardation, the following oligonucleotides were used: the
positive regulatory domain II (PRDII) sequence 5⬘-GGGAAATTCCGGGAA
ATTCC-3⬘, which contains two copies of the PRDII core element (1); the
PRDIV sequence 5⬘-AATGTAAATGACATAGGAAAACTGA-3⬘ (33); the
PRDIII-I sequence 5⬘-GAAAACTGAAAGGGAGAAGTGAAA-3⬘ (33); and FIG. 1. Selective IFN-␤ induction by TNF-␣ in nonmalignant
an NF-␬B sequence, 5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘, derived from HPV18-positive cells, showing intactness of the MAPK pathway. (A)
the immunoglobulin ␬ light-chain gene (44). The DNAs were synthesized in an RT-PCR products generated by IFN-␤- and GAPDH-specific primer
Applied Biosystems synthesizer using phosphoramitide chemistry and further sets (185 and 460 bp, respectively) were separated on 2% agarose gels.
purified by high-pressure liquid chromatography. For electrophoretic mobility The superimposed ethidium bromide-stained gel shows the quality of
shift assays (EMSAs), the annealed oligonucleotides were labeled with the RNAs used for the RT-PCR. The positions of the 18S and 28S
[␥-32P]ATP (Amersham; 3,000 Ci/mmol) with T4 polynucleotide kinase and gel rRNAs are indicated. Nonmalignant 444 hybrids, their tumorigenic
purified from a 15% polyacrylamide gel. Cellular extracts were prepared as segregants (CGL3 cells), and parental HeLa cells were incubated with
described previously (54), with the only modification being that N,N-(L-3-trans- 10 ng of TNF-␣ per ml for different periods of time. Lanes (⫺),
carboxyoxirane-2-carbonyl)-L-leucyl-agmatine (E64), 4-(2-aminoethyl)-benzol- untreated controls. (B) Western blot analysis of p38 MAPK. Twenty
sulfonylfluoride (Pefabloc SC), 1 mM NaF, and 0.2 mM Na3VO4 were included micrograms of total cellular extract per lane was separated in two
as protease inhibitors in concentrations suggested by the manufacturer (Roche). identical SDS–10% polyacrylamide gels. After transfer, the filters were
The protein concentration was determined by the Bradford method (Bio-Rad) incubated with either a phosphorylation-specific (Phospho-p38MAPK)
using defined amounts of bovine serum albumin as standards. The binding of or a phosphorylation-unspecific (p38MAPK) p38 antibody. Equal load-
NF-␬B, activation factor-2 (ATF-2), and c-Jun was performed in a 20-␮l reaction ing and protein transfer were confirmed by incubating the upper filter
volume containing 10% glycerol, 12 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH with an actin-specific antibody. The molecular masses are indicated.
7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.6 mg of bovine serum
albumin per ml, 2.0 ␮g of poly(dI-dC), and 2 to 4 ␮g of nuclear extract. After 5
min, 10,000 cpm of the [␥-32P]ATP 5⬘-end-labeled double-stranded oligonucle-
otide probe was added, and the incubation was continued for an additional 30 an IRF-1 antibody specific for the C-terminal region of human IRF-1 (sc-497x),
min at room temperature (58). EMSAs for the IRF family were performed in an IRF-2 antibody specific for the carboxy terminus of human IRF-2 (sc-498x),
20-␮l reaction volumes containing 10% glycerol, 70 mM Tris (pH 7.5), 30 mM Na an IRF-3 antibody with an epitope corresponding to full-length IRF-3 of human
acetate, 10 mM EDTA, 1 ␮g of poly(dI-dC), and 5 ␮g of nuclear extract. After origin (sc-9082x), and an IRF-7 antibody which recognizes amino acids 1 to 246
5 min, 10,000 cpm of the [␥-32P]ATP 5⬘-end-labeled double-stranded oligonu- of human IRF-7 (sc-9083x). Antibody to p48 (ISGF3␥) was from Transduction
cleotide probe was added, and the incubation was continued for an additional 15 Laboratories. The DNA-protein complexes were resolved on 5.5% nondenatur-
min at 4°C (7a). The sequence specificity of the binding was routinely controlled ing polyacrylamide gels (29:1 cross-linking ratio), dried, and exposed overnight to
in competition experiments by the addition of a 100-fold molar excess of either Fuji medical X-ray films.
unlabeled homologous or heterologous oligonucleotides. For monitoring the SDS-polyacrylamide gel electrophoresis and Western blotting. The same ex-
c-Jun and ATF-2 composition in supershift assays, 2 ␮g of polyclonal antibodies tracts (25 to 50 ␮g) used for the band-shift analyses were separated in 8 to 10%
directed against c-Jun or ATF-2 was added and the reaction mixture was further SDS-polyacrylamide gels, electrotransferred to polyvinylidene difluoride mem-
incubated for 1 h at 4°C. Selective binding of different IRF members was ana- branes (Immobilon-P; Millipore Corporation, Bedford, Mass.), and probed with
lyzed by adding 2 ␮g of poly- or monoclonal antibodies to the reaction mixture antibodies to IRF-1 (sc-497x; Santa Cruz), IRF-2 (sc-498x; Santa Cruz), and p48
prior to oligonucleotide supplementation. Specifically, the following antibodies (Transduction Laboratories). The incubation was carried out overnight in Tris-
(all obtained from Santa Cruz Biotechnology as TransCruz supershift reagents) buffered saline supplemented with 5% skim milk powder (Merck), 0.05% Tween
were used: a c-Jun antibody which recognizes both the nonphosphorylated and 20 (Sigma), and a 1:5,000 (IRF-1 and IRF-2) or 1:500 (p48) dilution of antibod-
phosphorylated forms of c-Jun (epitope corresponding to amino acids 56 to 69 ies. p38 and phospho-p38 mitogen-activated protein kinase (MAPK) were di-
mapping within the amino-terminal domain of the mouse c-Jun protein) (sc- rectly monitored in total cellular extracts obtained after lysis in SDS-polyacryl-
822x), an ATF-2 antibody specific for Thr-71-phosphorylated ATF-2 (sc-8398x), amide gel electrophoresis buffer (60 mM Tris, 2% SDS, 10% glycerol, 50 mM
VOL. 76, 2002 ABSENCE OF IFN-␤ EXPRESSION IN CERVICAL CARCINOMA CELLS
FIG. 2. TNF-␣ confers selective protection against EMCV infec-
tion in nontumorigenic HPV18-positive cells. Malignant and nonma-
lignant cells grown in microtiter plates were first pretreated for 24 h
with serial dilutions (1:2, indicated by the arrow) of TNF-␣ (10, 5, and
2.5 ng/ml) and infected with EMCV at an MOI of 0.1 as described in
Materials and Methods. To monitor the CPE, the cells were fixed and
stained with crystal violet. cc, uninfected control cells; vc, EMCV-
infected cells without prior TNF-␣ addition; Tc, TNF-␣ treated cells
not infected with EMCV. (A to C) 444, HeLa, and CGL3 cells, re-
spectively. (D) Quantification of the CPE after staining with crystal
violet. The dye was eluted, and its absorbance at 570 nm was deter-
mined spectroscopically. The bars indicate the percentages of pro-
283
dithiothreitol, 0.1% bromophenol blue, pH 6.8). Antibody dilutions were used as
recommended by the supplier (New England Biolabs, Frankfurt, Germany). The
bands were visualized with an anti-rabbit immunoglobulin G antibody conju-
gated with horseradish peroxidase using the ECL detection system (Amersham).
Equal protein transfer and loading were routinely controlled by reincubating the
filters with a monoclonal actin-specific antibody (ICN Biomedicals). For reincu-
bation with additional antibodies, the filters were stripped with 200 mM NaOH
for 5 min at room temperature.
RESULTS
Restoration of the TNF-␣-induced antiviral response in
nonmalignant HPV18-positive somatic cell hybrids: reexpres-
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sion of the IFN-␤ gene. To monitor the antiviral effect of
TNF-␣ on the IFN-␣/␤-regulatory pathway, we used HPV18-
positive HeLa cells and derived somatic cell hybrids as an
experimental model system (61). Tumorigenic HeLa cells can
be reverted to nonmalignant growth when fused to primary
human fibroblasts (designated 444 cells). Long-term in vitro
cultivation, however, leads to rare tumorigenic segregants of
the same hybrids (referred to as CGL3 cells), which permits
the investigation of cytokine signaling in a cellular environ-
ment harboring identical transcription cassettes of HPV18 (49)
but where the viral oncoproteins E6 and E7 are expressed in a
different genetic background. When such cells were treated
with TNF-␣, IFN-␤ mRNA could be detected as a 185-bp
RT-PCR fragment in nonmalignant hybrids (444 cells) which
became discernible as saturated quantities 4 h after TNF-␣
application and persisted for at least 24 h. When the same
experiment was carried out with RNAs obtained from malig-
nant segregants (CGL3 cells) or with parental HeLa cells, no
IFN-␤ expression occurred (Fig. 1A). The selective inducibility
of the IFN-␤ gene could not be attributed to a disturbance of
the TNF-␣ signal response or receptor engagement in malig-
nant cells, because p38MAPK MAPK phosphorylation, a known
hallmark for functional proinflammatory cytokine signaling
(65), became visible and disappeared with approximately the
same kinetics in all three cell lines without quantitative
changes of the net amount of nonphosphorylated p38 (Fig.
1B). Identical loading and protein transfer after Western blot-
ting were assessed by reincubating the same filter with an
actin-specific antibody.
To verify that the IFN-␤-specific mRNA was translated into
functional protein, a biological assay based on the susceptibil-
ity of nonresponsive cells to lysis after infection with EMCV
was used (37, 66) (Fig. 2). After being seeded in 96-well plates,
the cells were first preincubated with different amounts of
TNF-␣ ranging from 10 to 2.5 ng/ml. Only 444 cells were
protected (Fig. 2A), while both HeLa cells (Fig. 2B) and the
CGL3 tumorigenic segregants (Fig. 2C) were completely sen-
sitive to EMCV-mediated cell lysis, even at higher TNF-␣
tected cells relative to the untreated and the TNF-␣-incubated cell
controls (cc and Tc, respectively). (E) Determination of IFN-␤ syn-
thesis in 444 cells. Supernatants (in twofold serial dilutions) were
added to A549 indicator cells, and antiviral activity was determined as
described in Materials and Methods. The amounts of IFN-␤ produced
at different periods of time (4, 8, and 16 h) after treatment with 10 ng
of TNF-␣ per ml are expressed as international units per milliliter. All
standard deviations are given for three independent experiments per-
formed in triplicate.
284 BACHMANN ET AL. J. VIROL.

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FIG. 3. EMCV protection is mediated by IFN-␤. 444 cells were
pretreated with different concentrations of TNF-␣ prior to EMCV
infection in the presence of antibodies sufficient to neutralize IFN-␣
(␣), IFN-␤ (␤), or both (␣ ⫹ ␤) as described in Materials and Methods.
cc, uninfected control cells; vc, EMCV-infected cells without prior
TNF-␣ addition; Tc, TNF-␣-treated cells not infected with EMCV.

concentrations. These data illustrate that the antiviral activity

of TNF-␣ was reconstituted in nonmalignant hybrids even in
the presence of the viral oncoproteins but was lost in tumori-
genic segregants or parental HeLa cells. To estimate how
much IFN-␤ was definitively synthesized, the supernatants of
nonmalignant hybrids were removed and tested for IFN-␤ ac-
tivity against EMCV on A549 indicator cells, where TNF-␣
itself had no antiviral activity (66). Concordant with the RT-
PCR data (Fig. 1A), biologically active IFN-␤ was first detect-
able after 4 h, leading to an average accumulation of IFN-␤
ranging between 18 and 33 IU at 16 h after TNF-␣ adminis-
tration (Fig. 2E). To ensure that endogenous IFN-␤ synthesis
was responsible for the protective effect against EMCV infec-
tion, the same assay was performed in the presence of neutral-
izing IFN-␤ antibodies added 1 h prior to TNF-␣ supplemen-
tation. Figure 3 shows that only the addition of antibodies
against IFN-␤, and not addition of an IFN-␣-specific anti-
serum, significantly inhibited the TNF-␣-induced antiviral ac-
tivity. This supports the notion that IFN-␤ was in fact the key
effector protein which selectively protected nontumorigenic
cells against EMCV infection.
Exogenous IFN-␤ supplementation protects HPV-positive
cells from EMCV infection independently from the in vivo
phenotype. Since it has been reported that the expression of
viral oncogenes can interfere with IFN signaling (for a review,
see reference 30), it was mandatory to examine whether ma-
lignant and nonmalignant cells still reacted selectively when
IFN-␤ was added exogenously. In this case, all cell lines could
be protected against EMVC-mediated cytolysis independently
of whether IFN-␤ (Fig. 4) or IFN-␣ or IFN-␥ (A. Bachmann et
al., unpublished observation) was added to the tissue culture
medium. Note that protection was successful even after appli-
cation of less than 25 U of IFN-␤ per ml, which was in agree-
ment with our preceding data measuring the bioavailability of
444 cell-secreted IFN-␤ at 16 h after TNF-␣ addition (compare
Fig. 2E and 4A). Contrary to previous observations (3, 32, 42,
48), the present data unambiguously demonstrate that, as far
as the antiviral activity is concerned, both IFN-␣/␤ and IFN-␥
can still protect HPV18-positive cells against EMCV infection
(Fig. 4D), even after ongoing oncogene expression (see Dis-
cussion).
FIG. 4. Overall protection of HPV18-positive cells after exogenous
IFN-␤ supplementation. Malignant and nonmalignant cells grown in
microtiter plates were first pretreated for 24 h with serial dilutions (1:2,
indicated by the arrow) of IFN-␤ (100, 50, and 25 U) and infected with
EMCV at an MOI of 0.1 as described for Fig. 2. (A to C) 444, HeLa,
and CGL3 cells, respectively. (D) Quantification of the degree of
protection. The bars indicate the percentages of IFN-␤-protected cells
relative to untreated and IFN-␤-incubated control cells used as a
reference (indicated as cc and Tc, respectively). Standard deviations
are given for three independent experiments performed in triplicate.
IRF-1 and p48 (ISGF3␥) are selectively induced in nonma-
lignant cells upon TNF-␣ treatment. To gain further insight
into the differential regulation of the IFN-␤ gene in nonma-
lignant hybrids, we next examined the transcription factors
involved in IFN-␤ induction. One major key regulatory factor
which is activated after addition of proinflammatory cytokines
is NF-␬B (31). NF-␬B binds to the IFN-␤ promoter as a part of
a coordinately assembled multiprotein complex called the en-
hanceosome (for a review, see reference 36). At the DNA
level, the IFN-␤ upstream region is composed of four PRDs
(PRDI to -IV) which act synergistically in stimulating tran-
scription (17, 24, 36) (Fig. 5A shows a schematic overview).
Using parts of PRDII in comparison with an NF-␬B site ob-
tained from the immunoglobulin ␭ light-chain gene (44) for
EMSAs, both oligonucleotides showed approximately the
VOL. 76, 2002 ABSENCE OF IFN-␤ EXPRESSION IN CERVICAL CARCINOMA CELLS 285

same affinity in all three cell lines investigated. NF-␬B binding

already became discernible 30 min after TNF-␣ addition (Fig.
5B), which makes it unlikely that the absence of IFN-␤ induc-
ibility was due to a failure in cytokine signaling towards the
NF-␬B branch. When the same set of experiments were carried
out with PRDIV containing the cis-regulatory sequences for
c-Jun and ATF-2, a more complex binding pattern appeared.
When the origins and specificities of the various bands were
examined by addition of c-Jun (Fig. 5C) and phosphorylation-
specific ATF-2 (Fig. 5D) antibodies in supershift EMSAs, both
transcription factors again revealed roughly the same binding
kinetics.

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A completely different picture emerged when we monitored
the expression of IRF-1, which was originally identified as a
critical mediator of the IFN response (for a review, see refer-
ence 62). As depicted in Fig. 6A, TNF-␣ treatment led to a
strong induction of IRF-1 transcription exclusively in nontu-
morigenic hybrids (444 cells), whereas the gene was only mar-
ginally elevated in the malignant cells (CGL3 and HeLa cells).
The effect became even more pronounced when IRF-1 expres-
sion was examined by Western blot analysis (Fig. 6B). When
EMSAs were performed with oligonucleotides encompassing
the PRDIII-I region (Fig. 5A), significant IRF-1 binding was
obtained only with nuclear extracts derived from TNF-␣-
treated 444 cells (Fig. 6C, left). The authenticity of IRF-1 was
verified after addition of a specific antibody, which leads to a
disappearance of the induced band. IRF-3, IRF-7, and p48 do
not bind at this region after short-term TNF-␣ application (4
h), since the binding pattern was not affected after addition of
the respective antibodies (Fig. 6C, right). Conversely, consis-
tent with the EMCV infection assays described above (Fig. 4),
IRF-1 could be activated independent of the cell phenotype
when either IFN-␤ or IFN-␥ was exogenously supplemented
(Fig. 6D and E, respectively). No selective induction or over-
expression in tumorigenic cells was seen in analysis of the
expression of IRF-2 (Fig. 7A), a transcription factor which
antagonizes the function of IRF-1 by competing for the same
binding site (PRDI and -II) (for a schematic overview, see Fig.
5A) (62). The same was true for IRF-3 (2), encoded by a
transcriptional activator gene, which was constitutively tran-
scribed and not further stimulated after TNF-␣ application
(Fig. 7B).
p48 (ISGF3␥) is another IRF family transcription factor and
is the major binding component of ISFG3 (a multimeric com-
plex between STAT1 and -2) (5). Although p48 (ISGF3␥)
seems to be more involved in the autocrine amplification of the
so-called delayed IFN-␣/␤ response (24), knockout experi-

FIG. 5. EMSAs of NF-␬B, c-Jun, and ATF-2 at the IFN-␤ up-

stream regulatory region. (A) Schematic overview of the IFN-␤-regu-
latory region (enhanceosome). I to IV, PRDs and the relative binding
positions of c-Jun; ATF-2; IRF-1, -2, -3, and -7; and NF-␬B (p50/p65).
(B to D) HeLa, 444, and CGL3 cells were treated with TNF-␣ for 30
min and 4 h. Lanes (⫺), untreated controls. Nuclear extracts were
prepared for EMSAs with a PRDII and NF-␬B probe (B) or an
oligonucleotide probe harboring PRDIV (C and D). PRDIV EMSAs
were performed either in the absence (⫺) or in the presence (⫹) of
supershift antibodies directed against c-Jun (C) or phosphorylated
ATF-2 (D). Specific bands are marked by an arrow. Asterisks indicate
nonspecific binding.
286 BACHMANN ET AL.
FIG. 6. Selective IRF-1 induction by TNF-␣ in nonmalignant
HPV18-positive cells. (A) Transcriptional analysis of IRF-1 after
TNF-␣ treatment for 0.5, 1, 2, 4, and 8 h. Total RNA (5 ␮g/lane) was
separated on 1% agarose gels. Filters were consecutively hybridized
with probes specific for IRF-1 and ␤-actin. IRF-1 was exposed to
Kodak Biomax film and ␤-actin was exposed to Kodak X-Omat film for
1 day. The positions of the 18S and 28S rRNAs are indicated. (B)
Western blot analysis of nuclear extracts (50 ␮g/lane) after TNF-␣
application. After electrotransfer, the filters were incubated with an-
tibodies directed against IRF-1. Equal loading was assessed with an
actin-specific antibody. (C) Left, EMSAs using an oligonucleotide
probe harboring PRDIII-I. HeLa and 444 cells were treated with
J. VIROL.
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FIG. 7. IRF-2 and IRF-3 expression in HPV18-positive cells. (A)
IRF-2 Western blot analysis of nuclear extracts (50 ␮g of protein/lane)
of each cell line treated with TNF-␣ as indicated. After electrotransfer,
the same filters were consecutively incubated with antibodies raised
against IRF-2 and actin. (B) RT-PCR products of IRF-3 and GAPDH
(399 and 460 bp, respectively) after separation on 2% agarose gels. The
quality of the RNAs used for the RT-PCR is shown at the top. The
positions of the 18S and 28S rRNAs are indicated. Cells were incu-
bated for 16 h in the presence of 10 ng of TNF-␣ per ml. Lanes (⫺),
untreated controls.
ments have revealed that p48⫺/⫺ mice were impaired in their
antiviral activity against EMCV and VSV infections (27). To
assess the role of p48 (ISGF3␥) in the perturbation of TNF-
␣-mediated IFN signaling in our experimental system, p48
expression was examined. As depicted in Fig. 8A, only nonma-
lignant cells revealed significant p48 (ISGF3␥) mRNA levels at
4 and 8 h after TNF-␣ treatment, while the gene remained
transcriptionally silent in the malignant counterparts. Since it
has been reported that E7 of HPV16 can potentially interfere
with p48 (ISGF3␥) translocation into the nucleus (3), cells
were fractionated and the nuclear extracts were monitored by
Western blot analysis. To exclude cytoplasmic contamination,
the quality of nucleus-cytoplasm separation was controlled by
incubation of the filter with a cytoplasmic (pyruvate kinase
M2) marker protein (Bachmann et al., unpublished data) (34).
Similar to the case for IRF-1 (Fig. 6B), nuclear p48 (ISGF3␥)
accumulation occurred only in nonmalignant cells (Fig. 8B),
while gene expression was inducible with similar kinetics in all
three cell line when IFN-␤ or IFN-␥ was administered (Fig.
8C).
Complementation of a nontumorigenic phenotype between
different cervical carcinoma cell line restores antiviral activity.
TNF-␣ for 1, 4, and 16 h. Lanes (⫺), untreated controls. Right, PRD
III-I EMSAs with TNF-␣-treated (4 h) 444 extracts in the presence of
supershift antibodies directed against IRF-1, -2, -3, and -7 and p48.
The specific IRF-1 and supershift bands are indicated by arrows. (D
and E) Same as panel A but after treatment with IFN-␤ (10 U/ml) or
with IFN-␥ (10 U/ml). The IFN application was extended to 16 h.
VOL. 76, 2002 ABSENCE OF IFN-␤ EXPRESSION IN CERVICAL CARCINOMA CELLS
FIG. 8. Selective p48 (ISGF3␥) induction by TNF-␣ in nonmalig-
nant HPV18-positive cells. (A) Transcriptional analysis of p48 after
TNF-␣ treatment for 0.5, 1, 2, 4, and 8 h. Total RNA (5 ␮g/lane) was
separated on 1% agarose gels. Filters were consecutively hybridized
with probes specific for p48 (ISGF3␥) and ␤-actin. Exposure was done
on Kodak Biomax films (p48) or Kodak X-Omat films (␤-actin) for 3
days and 1 day, respectively. The positions of the 18S and 28S rRNAs
are indicated. (B) Western blot analysis of nuclear extracts (25 ␮g/
lane) at different times after TNF-␣ application. After electrotransfer,
the filters were incubated with a polyclonal p48 antibody. Equal load-
ing was confirmed with an actin-specific antibody. (C) Same as panel A
but after treatment with IFN-␣ (10 U/ml). IFN-␣ application was
extended to 16 h.
Based on the preceding experiments, we reasoned that cervical
carcinoma cells in general may lack TNF-␣-induced antiviral
activity, which should correlate with IRF dysregulation and
tumorigenicity. To test this prediction, HPV16-positive CaSki
cells and HPV18-positive SW756 cells were seeded in 96-well
plates and then tested in EMCV and VSV infection assays.
Both the CaSki and SW756 malignant cell lines were unable to
resist viral infection after pretreatment with TNF-␣ (Fig. 9A,
upper panels). As shown by RT-PCR analysis, deficiency of
viral resistance was again paralleled by an absence of IFN-␤
expression (Fig. 9B). Conversely, as already shown for HeLa
cells (Fig. 3), protection could be achieved when either IFN-
␣/␤ or IFN-␥ was exogenously supplemented, which was again
paralleled by IRF-1 and p48 expression (Bachmann et al.,
unpublished observations). When other malignant cells, such
as tumorigenic variants of in vitro-immortalized HPV16-posi-
tive human keratinocytes (HPK Ia cells) (10) were tested,
these cells were also found to be highly sensitive to EMCV and
VSV infection (Table 1). Only HPV16-positive SiHa cervical
carcinoma cells, which had low tumorigenic potential in animal
287
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FIG. 9. Virus protection in nontumorigenic hybrids between cervi-
cal carcinoma cells. (A) CaSki, SW756, SW756 ⫻ HeLa, and CaSki ⫻
HeLa hybrids were first pretreated for 24 h with serial dilutions (1:2,
indicated by the arrow) of TNF-␣ (10, 5, and 2.5 ng/ml) and infected
with EMCV at an MOI of 0.1 as described for Fig. 2. CaSki ⫻ HeLa
hybrids were infected with VSV at an MOI of 0.2. cc, uninfected
control cells; vc, infected cells without prior TNF-␣ addition; Tc,
TNF-␣ treated cells not infected with virus. (B) RT-PCRs of RNAs
obtained from SW756, CaSki, and somatic cell hybrids using HeLa
cells as a fusion partner after treatment with TNF-␣ for 16 h. For
details, see the legend to Fig. 1A. Lanes (⫺), untreated controls.
experiments, were partially protected against EMCV infection
after TNF-␣ treatment (ranging between 30 to 40%), but these
cells were again completely sensitive when infected with VSV
(Table 1).
In a recent study we have demonstrated that tumorigenicity
of HeLa and CaSki cells can be entirely suppressed after so-
matic cell hybridization. In contrast, hybrid formation between
HeLa and SW756 resulted in cell clones which were still ma-
lignant after heterotransplanation into immunocompromised
animals (59). Utilizing this complementation system in the
context of TNF-␣-mediated IFN-␤ signaling, antiviral activity
and protection against both EMCV and VSV infection could
be completely restored in nonmalignant CaSki ⫻ HeLa hy-
288 BACHMANN ET AL. J. VIROL.

TABLE 1. Summary of results of EMCV and VSV protection

studies in relation to the proliferative phenotype
of the cells in nude mice
Cell linea Tumorigenicityb Protection by TNF-␣

HPV-18
444 ⫺ ⫹
␤-444 ⫺ ⫹
CGL3 ⫹ ⫺
HeLa ⫹ ⫺
SW756 ⫹ ⫺

HPV-16
SiHa ⫹ ⫹/⫺

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CaSki ⫹ ⫺
HPK Ia late passage ⫹ ⫺

Hybrids
CaSki ⫻ HeLa ⫺ ⫹c
SW756 ⫻ HeLa ⫹ ⫺
SiHa ⫻ HeLa ⫹ ⫺
a
HPV 16- or 18-positive cervical carcinoma cells and derived cell hybrids.
b
Formation of tumors after subcutanous inoculation of 107 cells in both flanks
of 6-week-old female nude mice (59).
c
CaSki ⫻ HeLa hybrids were susceptible only to VSV infections.

brids, while SW756 ⫻ HeLa hybrids remained sensitive to

EMCV infection after TNF-␣ treatment (Fig. 9A, lower pan-
els). When IRF-1 and p48 (ISGF3␥) transcription in these cell
lines was monitored by Northern blotting, both genes were
found to become significantly reinduced by TNF-␣ in nonma-
lignant CaSki ⫻ HeLa cells but not in SW756 ⫻ HeLa hybrids,
where only marginal transcription was detectable (Fig. 10).
IRF-1 and p48 (ISGF3␥) reexpression resulted in complete
restoration of IFN-␤ transcription, as confirmed by RT-PCR
analyses (Fig. 9B).
DISCUSSION
Extending our previous studies to unravel immunological
escape mechanisms during HPV-induced carcinogenesis (for a
review, see reference 52), we found that the inducibility of the
IFN-␤ gene by TNF-␣ was eliminated in tumorigenic HPV18-
positive HeLa cells but was reconstituted in nonmalignant
HeLa ⫻ fibroblast hybrids (Fig. 1). Restoration of IFN-␤ ex-
pression resulted in successful protection of cells against infec-
tion with EMCV or VSV (37) (Fig. 2). Furthermore, the fail-
ure of TNF-␣ to induce an effective antiviral response in 444
cells in the presence of neutralizing antibodies against IFN-␤
(but not against IFN-␣) confirmed a direct involvement of
autocrine secreted IFN-␤ as an antiviral mediator elicited by
TNF-␣ (Fig. 3). Of particular interest also was the fact that
malignant cells remained protected against virus when either
IFN-␣/␤ or IFN-␥ was directly supplemented into the tissue
culture medium (Fig. 4). These data provide compelling evi-
dence that IFN signaling and the induction of an antiviral
function operate equally well in all cell lines and independently
from the proliferative phenotype in immunocompromised an-
imals.
The observation that TNF-␣-mediated induction of IFN-␤
was restricted to nontumorigenic hybrids strongly suggests that
the cross talk between the TNF-␣ pathway towards transcrip-
tional activation of the IFN-␤ gene is disturbed in cervical
FIG. 10. Restoration of IRF-1 and p48 (ISGF3␥) expression in
nonmalignant CaSki ⫻ HeLa hybrids. (A and B) Transcriptional anal-
ysis of IRF-1 in CaSki (A) and SW756 (B) cells and the corresponding
HeLa hybrids after TNF-␣ treatment for 1, 4, and 8 h. Total RNA (5
␮g/lane) was separated on 1% agarose gels. Filters were consecutively
hybridized with probes specific for IRF-1 and ␤-actin. (C) Same as
panels A and B but after hybridization with a p48 (ISGF3␥)-specific
probe. Exposure was done on Kodak Biomax films (p48) or Kodak
X-Omat films (␤-actin) for 3 days and 1 day, respectively. Lanes (⫺),
untreated controls.
carcinoma cells. This may have considerable implications for
immune evasion processes during progression to cervical can-
cer (52, 67). TNF-␣ represents an important regulatory cyto-
kine with immunomodulatory and growth-inhibitory functions
in nonmalignant HPV-positive keratinocytes (35, 51). TNF-␣
both suppresses transcription of the viral E6 and E7 oncogenes
and induces the expression of MCP-1, exclusively in nontu-
morigenic cells (51). MCP-1 belongs to a superfamily of small
secretory proteins called chemokines (39, 47), which recruit
and activate mononuclear cells, the first line of defense against
viral infection (45). Activated macrophages in turn not only
secrete additional TNF-␣, thereby amplifying the cytokine re-
sponse, but also are capable of inducing IFN-␣/␤, which have
strong antiviral functions, in their target cells (23, 38). It should
be emphasized that IFN-␤ induction represents the earliest
antiviral response which occurs by an protein synthesis-inde-
pendent pathway (for reviews, see references 17 and 19). Since
IFN-␣ is not able to be induced in cells lacking both copies of
the IFN-␤ gene (IFN-␤⫺/⫺ cells), it is thought that IFN-␤
VOL. 76, 2002 ABSENCE OF IFN-␤ EXPRESSION IN CERVICAL CARCINOMA CELLS
binding to its cognate receptor is a prerequisite for activation
of further IFN-␣ production (11). Consequently, loss of both
TNF-␣-mediated MCP-1 and IFN-␤ inducibility in tumori-
genic cells concomitant with the absence of a negative regula-
tory effect on viral E6 and E7 expression could provide an
explanation for the observed depletion of immunological ef-
fector cells in dysplastic lesions (22, 29, 46, 60, 63, 64), which
not only diminishes the immediate-early antiviral response but
also may increase the incidence of cervical cancer.
Although both E6 and E7 can counteract the function of
regulatory proteins involved in the ultimate IFN response (3,
40, 42, 48), it was amazing that tumorigenic cells still respond
to exogenous IFN treatment (Fig. 4). As reported recently,
HPV18 E6 affects IFN-␣ signaling by reducing Jak-STAT1/2
tyrosine phosphorylation in human fibrosarcoma cells (32).
Additionally, HPV16 E6 can bind to IRF-3, thereby impairing
Sendai virus-induced activation of IFN-␤ and 2⬘,5⬘-oligoadeny-
late synthetase transcription (48). However, it must be stressed
that both processes were not completely perturbed by E6,
strongly indicating that, at least in the latter case, other factors
(such as IRF-1 [see below]) can functionally substitute for
IRF-3. Nonetheless, because TNF-␣ can selectively suppress
HPV transcription in nontumorigenic cells (51), it was still
conceivable that the reduction of oncogene expression to
threshold levels may partially allow an IFN-␤ response. To
clarify this point, we used modified nontumorigenic HeLa ⫻
fibroblast hybrids which were additionally transfected with an
HPV18 E6-E7 transcription cassette under the control of the
␤-actin promoter. In those transfectants, only endogenous
transcription, and not the ␤-actin-driven E6-E7 transcription,
became suppressed upon cytokine treatment. Nevertheless,
TNF-␣-treated ␤-actin 444 cells were still protected against
EMCV infection, arguing against a direct involvement of viral
oncogene expression in the outcome of the antiviral response
(Bachmann et al., unpublished results).
By compiling the results of experiments which monitor tran-
scription factors engaged in the differential regulation of
IFN-␤ gene expression (36), the following picture emerged.
Using duplicated parts of PRDII in comparison to an NF-␬B
binding site derived from the immunoglobulin ␬ light chain
(44), TNF-␣ addition resulted in similar binding patterns when
nuclear extracts were analyzed in EMSAs (Fig. 5B). Therefore,
the absence of IFN-␤ inducibility in tumorigenic cells cannot
be attributed to inefficient cytokine signaling towards NF-␬B
activation, since no obvious differences in affinity and binding
kinetics could be discerned. An equivalent situation was found
with PRDIV-derived oligonucleotides harboring the recogni-
tion sequences for c-Jun and ATF-2. Binding of the latter to
PRDIV was paralleled by a threonine-specific phosphorylation
at position 71 (Fig. 5B), which occurred in the same temporal
relationship as detected for the phosphorylation of the up-
stream MAPK p38MAPK (Fig. 1B). p38MAPK represents a ma-
jor effector MAPK of ATF-2, which becomes transiently acti-
vated after addition of proinflammatory cytokines such as
TNF-␣ (65).
Regarding IRF-1, however, strong inducibility and DNA
binding to PRDIII-I after TNF-␣ treatment was achieved only
in nonmalignant cells, while the corresponding protein was not
detectable or was barely detectable in the tumorigenic coun-
terparts (Fig. 6A, B, and C). The reason for this discrepancy in
289
gene regulation is presently not understood. Suppression or
inefficient expression of IRF-1, however, can be epigenetically
modified by different degrees of chromatin condensation (55).
This notion was reinforced by a recent study demonstrating
that HPV16 E7 can recruit to the IFN-␤ promoter region a
histone deacetylase which blocks IRF-1 trans activation on
corresponding reporter constructs (42). Whether an altered
nucleosomal organization may account for inefficient IRF-1
transcription upon TNF-␣ treatment remains to be elucidated.
In any case, IRF-1 dysregulation in tumorigenic HPV-positive
cells might be of potential biological interest, especially in light
of the fact that IRF-1 can act as a tumor suppressor under
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specific conditions (for a review, see reference 62). Since over-
expression of IRF-1 induces apoptosis (62), it will be worth-
while in further studies to test whether the ectopic expression
of a dominant-negative mutant of IRF-1 alters the growth
properties of 444 cells towards malignancy in nude mice.
IRF-1 is functionally counteracted by IRF-2 by competition
for the same binding site within the IFN-␤ promoter (21).
Moreover, IRF-2 has a considerably longer half-life than
IRF-1 (approximately 8 h versus 30 min) (62) and is present in
uninduced cells [Fig. 7A, lanes (⫺)], probably to prevent un-
controlled IFN-␤ synthesis. As further outlined in Fig. 7A,
IRF-2 remained constitutively expressed throughout the cell
cycle, and no obvious quantitative difference in the protein
amount could be seen when extracts from malignant and non-
malignant cells were compared. An analogous situation was
found for the transcription of the IRF-3 gene, whose expres-
sion was also not further augmented after TNF-␣ addition
(Fig. 7B). Conversely, like the case for IRF-1, only nonmalig-
nant cells retained their ability to selectively synthesize p48
(ISGF3␥) (Fig. 8A). p48 (ISGF3␥) represents the DNA bind-
ing component of a trimeric complex (ISGF3) which induces,
jointly with STAT1 and STAT2, the transcription of antiviral
genes such as those for the 2⬘,5⬘-oligoadenylate synthetase or
double-stranded RNA-dependent protein kinase R (5). Al-
though p48 (ISGF3␥) is structurally related to IRF-1 and binds
to the IFN-␤ promoter (24), the protein does not execute a
redundant function in the cell but rather complements IRF-1
in inducing both IFN-␣/␤ and IFN-␥ responses (21). The pref-
erential accumulation of p48 (ISGF3␥) in the nuclear fraction
of 444 cells (Fig. 8B) is in contrast to recent results showing
that HPV16 E7 can block p48 (ISGF3␥) translocation into the
nucleus in spontaneously immortalized human keratinocytes
(HaCaT cells) after IFN-␣ treatment (3). Since this study in-
vestigated the role of E7 in IFN-␣ signaling in cells not asso-
ciated with a natural HPV infection, it is likely that the out-
come of the response may reflect the biological properties of
the respective model system, the dosage of the transduced
exogenous viral oncogene, the nature of the exogenous stim-
ulus, and/or the phenotype of the host cell.
Another important aspect of our analysis is the correlation
between nontumorigenicity and the antiviral activity induced
by TNF-␣. Considering our preceding experiments, we realized
that HeLa ⫻ fibroblast hybrids, which were converted to ma-
lignancy via ectopic c-fos expression (58), have almost com-
pletely lost their ability to block EMCV cytolysis after TNF-␣
addition (Bachmann et al., unpublished observations). We
therefore went on to use an additional cell system which is
based on the fact that fusion of two malignant cells results in
290 BACHMANN ET AL.
nonmalignant hybrids when different tumor suppressor genes
are affected. Complementation to nontumorigenicity cannot
be accomplished when the same gene or pathway is defective
(7, 43). Accordingly, when HPV18-positive HeLa cells or
HPV16-positive CaSki cells, both of which are highly suscep-
tible to viral infection after pretreatment with TNF-␣ (Fig. 9A,
upper panels), were fused, the resulting hybrids were nontu-
morigenic after inoculation into immunocompromised ani-
mals. In contrast, hybrid formation between HeLa cells and
TNF-␣-unresponsive HPV18-positive SW756 cervical carci-
noma cells (Fig. 9A, lower panels) failed to suppress tumor
formation (59). When those hybrid clones were challenged
with viral infection after TNF-␣ treatment, antiviral activity
could be completely restored in nonmalignant CaSki ⫻ HeLa
hybrids, whereas SW756 ⫻ HeLa hybrids (and SiHa ⫻ HeLa
hybrids) remained sensitive (Fig. 9A; Table 1). Reconstitution
of a functional antiviral response was therefore not a peculiar-
ity of the initially utilized HeLa ⫻ fibroblast hybrid system but
rather correlated with nonmalignancy and the capability to
reexpress IRF-1 and p48 (ISGF3␥) (Fig. 10) and, in turn,
IFN-␤ (Fig. 9B). This supports the notion that the antiviral
response of TNF-␣ is determined by the in vivo phenotype of
the respective HPV-positive host cell line rather than by on-
cogene expression per se.
Besides immunostimulatory and antiviral activities, the re-
striction of endogenous IFN-␤ production to nontumorigenic
cells also involves an additional interesting feature which may
explain the long latency period between viral infection and the
final progression to cervical cancer (67, 68). IFN-␤ synthesis
inversely correlates with angiogenesis as well as with cell pro-
liferation and, under some circumstances, with terminal differ-
entiation (4). Notably, IFN-␤ can down-regulate angiogenic
factors such as basic fibroblast growth factor (56), interleukin-8
(57), and matrix metalloproteases (16), all of which are neces-
sary to promote tumor growth and metastasis. Furthermore,
upon analysis of human keratinocytes in medium either favor-
ing or preventing terminal differentiation, IFN-␤ expression
was detectable only in cells without proliferating cell nuclear
antigen (PCNA). Similar results were obtained from the im-
munohistological evaluation of normal tissue sections, where
IFN-␤ production was restricted to nondividing suprabasal cell
layers (4). It will therefore be the aim of further studies to
analyze these additional properties of IFN-␤ in the context of
HPV-induced carcinogenesis in greater detail.
ACKNOWLEDGMENTS
We thank Eric Stanbridge (University of California, Irvine), Claudia
Denk (DKFZ, Heidelberg, Germany), and Matthias Dürst (University
of Jena, Jena, Germany) for providing cell lines. The technical help of
Anita Weyland is appreciated.
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