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Title:
ASSAY METHOD FOR IDENTIFYING INHIBITORS OF THE EXPRESSION OF THE ONCOPROTEINS E6 AND E7 OF THE HUMAN PAPILLOMA VIRUS (HPV), RECOMBINANT HOST CELL AND KIT FOR USE IN THE AFORESAID METHOD
Document Type and Number:
WIPO Patent Application WO/2006/045833
Kind Code:
A1
Abstract:
The invention concerns an epithelial host cell stably transfected with a recombinant nucleic acid construct comprising the nucleic acid sequence of a reporter gene, for example luciferase, operatively linked with a second nucleic acid sequence comprising the enhancer elements and the promoter of the genes coding for the HPV proteins E6 and E7. The host cell of the invention is used as a reporter system in a high yield cell assay for the identification of molecules useful in the treatment of tumours induced by papilloma virus. The invention also concerns a kit for performing the assay.

Inventors:
SANTO LANDOLFO (IT)
LEMBO DAVID (IT)
Application Number:
PCT/EP2005/055598
Publication Date:
May 04, 2006
Filing Date:
October 27, 2005
Export Citation:
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Assignee:
UNIV DEGLI STUDI TORINO (IT)
SANTO LANDOLFO (IT)
LEMBO DAVID (IT)
International Classes:
C12Q1/68
Domestic Patent References:
WO1999021979A11999-05-06
WO2003000279A12003-01-03
WO2003096988A22003-11-27
Foreign References:
US5484732A1996-01-16
Other References:
WHITE PETER W ET AL: "Inhibition of human papillomavirus DNA replication by small molecule antagonists of the E1-E2 protein interaction.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. 18 JUL 2003, vol. 278, no. 29, 18 July 2003 (2003-07-18), pages 26765 - 26772, XP002364330, ISSN: 0021-9258
BAO RUDI ET AL: "Use of a surrogate marker (human secreted alkaline phosphatase) to monitor in vivo tumor growth and anticancer drug efficacy in ovarian cancer xenografts", GYNECOLOGIC ONCOLOGY, vol. 78, no. 3 Part 1, September 2000 (2000-09-01), pages 373 - 379, XP002364442, ISSN: 0090-8258
HORLICK R A ET AL: "Combinatorial gene expression using multiple episomal vectors", GENE, ELSEVIER, AMSTERDAM, NL, vol. 243, no. 1-2, February 2000 (2000-02-01), pages 187 - 194, XP004187688, ISSN: 0378-1119
RORKE E A ET AL: "STABLE EXPRESSION OF TRANSFECTED HUMAN INVOLUCRIN GENE IN VARIOUS CELL TYPES EVIDENCE FOR IN-SITU CROSS-LINKING BY TYPE I AND TYPE II TRANSGLUTAMINASE", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 97, no. 3, 1991, pages 543 - 548, XP009060603, ISSN: 0022-202X
LEMBO DAVID ET AL: "A cell-based high-throughput assay for screening inhibitors of human papillomavirus-16 long control region activity", FASEB JOURNAL, vol. 19, no. 12, October 2005 (2005-10-01), XP002364331, ISSN: 0892-6638
Attorney, Agent or Firm:
Comoglio, Elena (TORINO, IT)
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Claims:
CLAIMS
1. An epithelial host cell containing one or more copies of a recombinant nucleic acid construct comprising the nucleic acid sequence of a reporter gene operatively linked with a second nucleic acid sequence comprising the enhancer elements and the promoter of the genes coding for the proteins E6 and E7 of HPV, the host cell being stably transfected with the recombinant nucleic acid construct.
2. The host cell according to Claim 1, wherein the second nucleic acid sequence is the LCR (Long Control Region) of HPV.
3. The host cell according to Claim 2, wherein HPV is a high risk HPV.
4. The host cell according to Claim 3, wherein HPV is selected among HPVI6 and HPV18.
5. The host cell according to any of Claims 1 to 4, wherein the reporter gene is the gene coding for the enzyme luciferase.
6. The host cell according to any of Claims 1 to 5, which is a keratinocyte.
7. The host cell according to any of Claims 1 to 6, which is a human cell.
8. A method for the identification of inhibitors of the expression of the proteins E6 and E7 of HPV, comprising the steps of: contacting a host cell as defined in any of Claims 1 to 7 with a molecule which is a candidate as an inhibitor of the expression of the proteins E6 and E7 of HPV; determining the activity of the reporter gene in the presence of the candidate molecule; comparing the activity of the reporter gene in the presence of the candidate molecule with the activity of the reporter gene in the absence of the candidate molecule, a decrease in the activity of the reporter gene in the presence of the candidate molecule being indicative of the ability of that molecule to inhibit the expression of the proteins E6 and E7 of HPV.
9. The method according to Claim 8, wherein a plurality of molecules is tested simultaneously, each one in a reaction vessel, preferably each one in one well of a microtitration plate.
10. A method according to Claim 8 or 9, wherein the candidate molecule is selected from the group consisting of synthetic and natural small molecules, nucleic acids, natural proteins and recombinant proteins .
11. A kit for the identification of inhibitors of the expression of the E6 and E7 proteins of HPV, comprising a culture of host cells as defined in any of Claims 1 to 7, as well as means and/or reagents for determining the activity of the reporter gene.
Description:
Assay method for identifying inhibitors of the expression of the oncoproteins E6 and E7 of the human papilloma virus (HPV) , recombinant host cell and kit for use in the aforesaid method

The invention concerns a high yield assay for the identification of inhibitors of the expression of the oncoproteins E6 and E7 of the human papilloma virus (HPV) , as well as a recombinant host cell suitable for use in the assay of the invention and a kit comprising such host cell.

The assay and the kit of the invention enable the rapid screening of banks of molecules for the purpose of selecting compounds capable of inhibiting the expression of the oncoproteins E6 and E7 of HPV - in particular of high risk HPV - in tumour cells. Given their ability to inhibit the expression of the oncoproteins of HPV, the substances thus selected can be inserted in programmes for the development of drugs for the treatment of tumours induced by HPV.

The high risk human papilloma viruses (for example HPV-16, HPV-I8, HPV-31 and HPV-33) represent the principal cause of cancer of the uterine cervix and are involved in the aetiology of tumours of other anatomical regions, such as for example the head and neck. Their genome is present in almost 100% of cases of carcinoma of the uterine cervix and in 20% of tumours of the pharyngo-oral cavity. Throughout the world, about 500,000 new cases of carcinoma of the uterine cervix are diagnosed every year and about one third of these result in the death of the patient. Prevention programmes based on the Papanicolau test (Pap test) have significantly reduced the incidence of this type of tumour in the more developed countries; nonetheless, carcinoma of the cervix still

represents one of the major causes of death in women in many parts of the world. Although various studies are in progress, no vaccine is yet available. Hence the only preventive strategy is based on early diagnosis and on the surgical removal of the dysplastic lesions. In spite of the fact that the main mechanisms involved in the aetiopathogenesis of the tumours caused by HPV have been adequately elucidated, no drugs are at present available for their treatment. The process of identification and development of anti-HPV drugs has until now been impeded by the technical difficulties of cultivating the virus in vitro, by the limited number of viral enzyme activities, which traditionally represent potential pharmacological targets, and by the lack of assays for the high yield screening of molecules with antiviral activity.

The object of the present invention is therefore to provide a high yield assay for the identification of molecules capable of inhibiting the fundamental event in the pathogenesis of tumours caused by high risk HPV, namely the constitutive expression of the early viral oncoproteins E6 and E7.

It is in fact known that during the process of cell transformation with leads to the development of a carcinoma, the DNA of the high risk HPV is integrated into the cell genome. Owing to this integration event, the viral genome is interrupted and its transcription programme is completely altered. As a result, constitutive expression of the oncoproteins E6 and E7, which are the only viral proteins which are expressed in the cells of the tumour, takes place. E6 and E7 are responsible for the triggering of the process of transformation of the infected cell in that by suppressing the function of the cellular anti-oncogenes p53 and Rb they

profoundly alter the control of cell proliferation. The transcription of the viral oncoproteins is controlled by a nucleic acid sequence about 1 kb long designated as LCR (Long Control Region) , whose activity is regulated by the binding of a number of cellular transcription factors. The LCR sequence contains the early promoter (for example, P 9 7 in HPV-16 and P 105 in HPV-18) and the enhancer elements necessary for controlling the expression of the oncoproteins E6 and E7.

Since a number of studies have shown that the continued expression of E6 and E7 is a necessary condition for the tumour cell to maintain the transformed phenotype, it is possible to conceive a therapeutic strategy devoted to suppressing the expression of those proteins. Indeed the proof of this principle has already been provided (DeFillipis et al., J. Virol., 77: 1551-1563, 2003; Butz et al., Oncogene, 22: 5938-5945, 2003) . Although these studies have demonstrated the theoretical validity of this approach, the techniques used (constitutive expression of an inhibitory protein and gene silencing with interfering RNA) are not transferable to clinical practice, where the use of drugs consisting of small molecules is preferable. Thanks to the recent development of innovative technologies in the field of the synthesis and purification of new chemical compounds, collections of hundreds of thousands of small molecules synthesized by combinatorial chemistry or purified from natural materials (plant or microbial) have become available, and represent an extremely important source for the identification of new pharmacological activities. However, in order to be able to analyze such a large number of molecules in time periods and at a cost compatible with the development of new drugs, it is essential to have available a rapid, economical and high yield assay.

Craigo et al. , Antiviral Res. 47: 19-28, 2000 describe a method for identifying molecules capable of inhibiting the expression of the genes coding for the oncoproteins of HPV based on the transient transfection of C33A cells with a recombinant plasmid comprising the gene of luciferase placed under the transcriptional control of the promoter of the LCR sequence of HPV-I6. The execution of this assay requires a series of technical operations which can be summarized as follows:

(i) Seeding of the cells (day 0) . The cells are seeded in a format which is not compatible with a high yield assay (for example 60 or 35 mm diameter plates) .

(ii) Transient transfection of the cells (day 1) with a recombinant plasmid comprising the gene for luciferase and the LCR sequence of HPV-16. This step requires the use of specific reagents which include, apart from the purified recombinant plasmid, also a purified normalization plasmid and the reagents necessary for the transfection. Furthermore, this step must be effected by an operator provided with specialized knowledge and skills.

(iii) Addition of the molecules to be tested (day 2) . (iv) Measuring of the luciferase specific activity (day 3) . For this purpose the culture medium is removed, the cells are washed and detached from the substrate, then the cells are centrifuged and the supernatant is removed. The cell sediment is lysed with a suitable lysis buffer. The cell lysates are again centrifuged to remove the subcellular components, and the sample thus obtained is subjected to the dosing of the protein content, for example with the method of Bradford. Then the luciferase activity and the activity of the reporter gene of the normalization plasmid (for example renilla luciferase or beta-galactosidase) are measured. The samples are read one at a time in a luminometer. The results obtained

with the dosing of the luciferase must be normalized with those obtained by measuring the activity of the indicator of the reporter plasmid, to eliminate differences between different samples due to possible variations in the transfection efficiency. To obtain the luciferase specific activity, the normalized luciferase activity is in turn divided by the concentration of the proteins obtained by the Bradford method.

It is apparent that the assay the test described above is not suitable for the screening of large number of molecules since it requires long time periods, it must be effected by specialized operators (the necessary skill represents a limit and increases the risk of errors) and above all it requires the repetition of the transfection operations each time, which results in the continuous need for purified recombinant plasmids and many reagents and commercial kits (for example plasmid purification kit, transfection kit, protein estimation kit, kit for estimation of luciferase activity and of the normalisation plasmid, etc.) . Moreover, the procedure described above is not practically applicable to a high yield assay format, namely a 96-well format. Consequently, this method makes it possible to test at the maximum a few tens of substances per week.

The present inventors have now succeeded in developing a high yield assay that enables the rapid identification of molecules capable of inhibiting the transcription activity of the LCR sequence of HPV and, consequently, the expression of the oncoproteins E6 and E7 of the virus.

In particular, the present inventors have succeeded in stably transfecting an epithelial host cell with a recombinant

nucleic acid construct comprising the nucleic acid sequence of a reporter gene operatively linked with a second nucleic acid sequence comprising the enhancer elements and the promoter of the genes coding for the HPV proteins E6 and E7, preferably an HPV LCR sequence.

This result is unexpected in view of the fact that, to the inventors' knowledge, no studies exist wherein an indicator plasmid containing an HPV LCR sequence operatively linked to an indicator gene is used in a stable manner, in other words is integrated into the genome of a host cell. It was not possible to predict a priori whether the approach could also function with the stably integrated plasmid and whether in that context the LCR sequence would be capable of maintaining its own responsivity to known inhibitory stimuli identified with a transient transfection approach.

Further, it was not possible to be reasonably certain of obtaining stable cell lines capable of expressing sufficient levels of activity of the indicator gene (e.g. luciferase activity) for the development of a miniaturized assay (that is an assay with 96-well microtitration plates) , which was the object of the present invention.

The stably transfected cells were in fact obtained several months after the first transfection event, in other words after selection in G418 for several weeks, a subsequent selection of individual clones by limit dilution which involved prolonged work and the characterization of tens of clones in order to obtain some with the required characteristics .

The host cell of the invention, being stably transfected, has

the advantage of being suitable for use in an assay, which in contrast to the assay described in the paper by Craigo et al. cited above, does not require the continued repetition of the transfection operations.

As used in the present specification, the expression "stably transfected host cell" indicates a cell capable of maintaining the reporter plasmid in episomal or integrated form for at least 100 days of continuous culture.

Hence the scope of the present invention includes an epithelial host cell containing one or more copies of a recombinant nucleic acid construct comprising the nucleic acid sequence of a reporter gene operatively linked with a second nucleic acid sequence comprising the enhancer elements and the promoter of the genes coding for the HPV proteins E6 and E7, the host cell being stably transfected with the recombinant nucleic acid construct.

The invention further includes a high yield assay for the identification of inhibitors of the expression of the HPV proteins E6 and E7, based on the use of the aforesaid stably transfected host cell. In particular, the assay of the invention comprises the steps of:

- contacting the aforesaid stably transfected epithelial host cell with a molecule which is a candidate as an inhibitor of the expression of the HPV proteins E6 and E7;

- determining the activity of the reporter gene in the presence of the candidate substance;

- comparing the activity of the reporter gene in the presence of the candidate molecule with the activity of the reporter gene in the absence of the candidate

molecule, the decrease in the activity of the reporter gene in the presence of the candidate molecule being indicative of the ability of that molecule to inhibit the expression of the HPV proteins E6 and E7.

Preferably, the second nucleic acid sequence of the recombinant nucleic acid construct is the LCR (Long Control Region) sequence of HPV. Still more preferably, this second nucleic acid sequence is the LCR sequence of a high risk human papilloma virus, such as for example HPV-I6 or HPV-I8.

Since the LCR sequence of HPV contains an enhancer element which is active only in the keratinocytes, it is preferable that the stably transfected epithelial host cell of the present invention be a keratinocyte. Still more preferably, immortalized or tumoral human keratinocyte lines which do not contain endogenous copies of the viral LCR are used for the stable transfection.

As indicated above, the host cell of the invention is transfected with a recombinant nucleic acid construct (for example recombinant DNA) which comprises a reporter gene operatively linked with promoter and enhancer sequences which control the expression of the oncogenes of HPV. Two nucleic acid sequences are "operatively linked" when there is a functional relationship between them. In the present case, the reporter gene is operatively linked with the HPV promoter and enhancer sequences when its expression is under the control of those sequences. Two operatively linked nucleic acid sequences do not necessarily have to be contiguous: they can be at a certain distance one from the other, provided that they maintain a functional relationship.

The reporter gene used in the present invention is any gene the expression whereof gives rise to an easily observable and detectable phenotype. For example, a reporter gene codes for an enzyme which acts on a substrate giving rise to a coloured, fluorescent or luminescent product. A preferred reporter gene for use in the present invention is the gene which codes for the luciferase enzyme of the firefly. Luciferase presents important advantages as a reporter system since this enzyme is not expressed by mammalian cells, has a short half-life and its activity is easily and rapidly detectable with commercially available kits. Other reporter genes suitable for use in the invention are for example chloramphenicol acetyl transferase (CAT) , beta galactosidase, alkaline phosphatase and green fluorescent protein (GFP) .

A preferred nucleic acid construct for use in the invention is a recombinant plasmid comprising the entire LCR sequence from nucleotide 7009 to nucleotide 124 of HPV-16 bound 5' with respect to the gene coding for the firefly luciferase. In this reporter system, the luciferase activity is in fact a proportional measure of the transcriptional activity of the LCR sequence. Consequently, a decrease in the luciferase activity determined in the presence of a candidate molecule as an inhibitor of the expression of the HPV oncoproteins indicates that that the molecule does indeed have inhibitory properties.

A kit for the screening of molecules capable of inhibiting the expression of the HPV proteins E6 and E7, comprising a stably transformed epithelial host cell as previously defined, means and/or reagents for determining the activity of the reporter gene also falls within the scope of the invention. Candidate molecules as inhibitors of the

expression of E6 and E7 proteins of HPV are commonly selected from the group consisting of small (i.e. low molecular weight) natural and synthetic molecules, nucleic as well as acids and natural and recombinant proteins. In a preferred form, the kit of the invention comprises a 96-well microtitration plate and reagents for the determination of the activity of the enzyme luciferase.

The invention here described markedly reduces the time necessary for the completion of a screening assay for inhibitors of the HPV oncoproteins. The assay of the invention can for example be effected according to the following time schedule:

1. Day 0: seeding of the cells in 96-well plates;

2. Day 1: addition of the molecules to be tested;

3. Day 2: addition of the luciferase activity revealing agent directly into the wells and simultaneous reading of all the wells on the plate in a luminometer.

The principal advantages of the assay of the invention compared to the known technology are listed below and are due to the use of a stably transfected host cell as the reporter system:

(i) No transfection event is necessary, which makes it possible to save at least one day of work and many hours of manipulation, as well as the plasmids and the reagents which would be required to repeat the transfection each time, (ii) There are no problems connected with possible differences in the transfection efficiency between the different samples. Hence it is not necessary to use a normalization system.

(iii) Through the use of the gene coding for luciferase as a reporter gene, sample preparation operations (such as for

example the removal of the medium, washing, harvesting and centrifugation of the cells, and the protein estimation) are not required, since a commercial kit is available (Steady-Glo assay system, Promega) which enables direct analysis in a 96- well format. The only operation required is the addition of the reagent directly into the well containing the cells without removing the culture medium. This operation can be performed with a multichannel pipetter. The reagent lyses the cells and simultaneously provides the substrate for the luciferase.

(iv) The luciferase activity can be simultaneously revealed in the 96 wells of the plate with a suitable luminometer, thus decreasing the analysis times.

Hence, the invention described makes it possible to test hundreds of molecules per day and thousands of molecules per week rapidly and at modest cost compared to the previous technology.

The invention finds its application in research and development programmes for drugs intended for the treatment of tumours induced by high risk human papilloma viruses and in particular in those programmes for screening of collections of molecules derived from combinatorial chemistry or extracted from plants or from microbial cultures.

The examples that follow are provided exclusively for illustrative purposes and are not in any way intended to limit the scope of the invention.

EXAMPLE 1

To obtain the epithelial cell line of the invention, about 20 μg of the plasmid described in Kammer et al. , (J. Gen. Virol.

2000, 81: 1975-1981) were transfected into the HaCaT cell line of immortalized human keratinocytes which allow a high transcriptional activity of the LCR sequence of HPV-16. To select the stably transfected cells, the plasmid pST-Neo (Katoh et al. , Cell Structure and Function 2: 575-580, 1987) which confers resistance to the antibiotic geneticin was cotransfected at 20 times lower doses. From the day following the transfection, the cells were cultured in a medium containing 400 μg/ml of geneticin. The cell clones obtained 3 weeks after the transfection were harvested and subcloned by the limit dilution technique. In this way, a large number of clones which express luciferase activity were isolated. For the development of the assay of the invention, the clone possessing the highest luciferase specific activity was selected. This clone was named P13. Polymerase chain reaction (PCR) analysis confirmed that the P13 clone indeed contained the LCR sequence of HPV-16. The clone multiplied adhering to the substrate and was cultured in a common culture medium consisting of Dulbecco modified Eagle medium, with the addition of 10% of foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and 200 μg/ml of geneticin.

EXAMPLE 2

In order to be able to use the P13 clone in the development of a standardized high yield assay, the following parameters were evaluated:

- the expression of luciferase activity with time (stability) ;

- the compatibility with the use of a high yield format (96- well plates) ;

- the response to molecules of known inhibitory activity

(validation) .

The stability of the clone was evaluated for a time period of 100 days, at intervals of 10 days, a mean luciferase activity of about 400 RLU/μg of protein being detected, which remained constant with time.

To evaluate the compatibility of the clone with the development of a high yield assay, in other words the use in a 96-well plate format, 5,000, 10,000 and 20,000 cells per well were seeded and the luciferase activity was evaluated. The mean values obtained were 1,700, 2,700 and 6,000 RLU respectively. Since the base value of the assay (parental cells or a sample with no proteins) is around 40 RLU, the aforesaid values are clearly compatible with the use of the assay in a high yield format.

To verify whether the LCR sequence had maintained a normal transcriptional control in the selected clone, the assay was subjected to a validation assay using two known inhibitory stimuli: the cytokines TGF-βl and TNF-α (Woodworth et al . , J. Virol. 64: 4767-4775, 1990; Baldwin et al. , J. Virol., 78: 3953-3964, 2004; Kyo et al . , Virology, 200: 130-139, 1994) . Treatment of the P13 clone with 100 ng/ml of the aforesaid cytokines for 24 hours inhibited the luciferase activity by 68% and 55% respectively. This result demonstrates that the LCR sequence integrated in the P13 clone has maintained a normal transcriptional control and hence the P13 clone can be used for the identification of inhibitory molecules.

The high yield identification of inhibitory molecules is effected by seeding 20,000 cells of the P13 clone per well in 96-well plates and adding a different molecule to each well.

The assay for luciferase activity effected 24 hours after the treatment will enable the identification of those molecules capable of inhibiting the activity of the LCR. This activity will be confirmed by the common cytotoxicity assays to exclude the possibility that the inhibition of the luciferase activity is due to a toxic effect of the molecule.

EXAMPLE 3

On day 0, the reporter cell line (for example the P13 clone) is seeded at a density of 20,000 cells per well into a 96- well plate. The cells are cultured in an 80 microlitre volume of medium and incubated at 37°C at a carbon dioxide pressure of 5% in a humid environment.

The following morning (day 1) , 20 microlitres per well of the dilutions of the different molecules to be tested are added (one molecule per individual well) . As control, a column of 8 wells treated exclusively with the diluent of the molecules is used. To transfer the molecules rapidly, a multichannel pipetter or an automated dispenser can be used. Incubate for 24 hours under the conditions described above.

On day 2, the luciferase activity is measured with the Steady-Glo Luciferase Assay System Kit (Promega) . The luminescence is measured in a luminometer capable of reading 96-well plates. The luminescence values of each treated well are compared with the mean of the values of the untreated wells, to identify inhibitory or stimulatory molecules.

EXAMPLE 4

Screening of a panel of cytokines using the HPV-16LCR reporter cell line (clone P13)

In order to exploit the cell-based assay, clone P13 was used as a reporter cell line to screen a panel of 32 cytokines belonging to different functional classes in a 24-well format. All cytokines were tested three times in duplicate at a concentration of 50 ng/ml; incubation time was 24 hours. After screening, the cytokines were classified into three groups according to the percent of LCR inhibition (Table 1) . Group I comprised the 21 cytokines that showed no or little inhibitory activity (0-29%) ; Group II comprised the 3 cytokines, namely Activin, IL-lβ and IFN-γ, that exerted a moderate inhibitory activity (30-49%) ; Group III comprised the 8 cytokines, namely IL-4, IL-I3, TGF-βl, TGF-β2, TGF-β3, TNF-α, IFN-α and IFN-β, that exerted a high inhibitory activity (50-70%) . No stimulatory cytokine was detected by the assay.

Table 1. Effect of cytokines on HPV-16LCR activity

% of LCR inhibition

Cytokines (mean values ± SD) Group

Anti-inflammatory

IL-4 56.6 ± 7.2 III

IL-10 0 I

IL-13 64.3 ± 5.8 III

TGF-βl 61.4 ± 8.4 III

TGF-β2 60.1 ± 6.5 III

TGF-β3 58.9 ± 5.9 III

Activin 32.4 ± 4.3 II

GDF-15 16.3 ± 2.1 I

Osteonectin 0.2 ± 0.03 I

Pro-inflammatory

IL-lβ 33.3 ± 4.6 II

IL-15 12.6 ± 1.8 I

IL-17 29.6 ± 3.6 I

IL-18 3 ± 0.5 I

IL-19 9 ± 1.2 I

IL-20 10.2 ± 0.8 I

IL-22 10.3 ± 1.7 I

TNF-α 53.7 ± 4.3 III

GM-CSF 12 + 0.7

Growth factors

IL-3 2.1 ± 0.09

IL-6 5.2 ± 0.3

IL-7 2.5 ± 0.5

IL-21 2.4 ± 0.4

Chemotactic factors

IL-8 0

IP-IO 5. 2 ± 0.9

LEC 16.1 ± 2.3

MIP-lα 0

MlP-lβ 27.3 ± 3.8

NAP-2 0

1-309 0.9 ± 0.2

Interferons

IFN-α 58.2 ± 7.6 II I

IFN-β 63.1 ± 4.8 II I

IFN-γ 35.5 ± 4.3 I I

Since the assay output depends partly on the cytostatic or cytotoxic activity of a given compound, each cytokine in Group III was tested on clone P13 by the MTT proliferation assay. None showed an antiproliferative activity at the concentration and time point selected for the luciferase assay (data not shown) . These findings indicate that the reduction in luciferase activity was not a consequence of reduced cell viability.

Among the cytokines of Group III, IL-4 and IL-13 are particularly interesting since these molecules have never been reported as inhibitors of LCR transcriptional activity. Thus, they were selected for a more detailed examination. In order to determine the dose of IL-4 and IL-13 which produces a 50% inhibition of LCR activity (ID 50 ), clone P13 was treated with a different concentrations of cytokines, ranging between 0.1 ng/ml and 250 ng/ml. Both cytokines reduced LCR activity in a dose-response fashion. Fig. 1 shows the results obtained. Specifically, Fig. 1 shows the dose-dependent

reduction of LCR-driven transcription obtained by seeding cells of clone P13 on 24-well plated and then treating them for 24 hours with increasing concentrations of IL-4 and IL- 13. The results represent the mean +SD of three similar experiments performed in duplicate.

Nonlinear regression data analysis calculated an ID 50 of 4 ng/ml for IL-4 and 11.3 ng/ml for IL-13.

In order to investigate possible interactions among cytokines that trigger different signalling pathways, co-stimulation experiments were performed. A cytokine concentration level of 5ng/ml for IL-4 and TGF-βl and 100U/ml for IFN-α and IFN-γ was selected. These doses produce approximately 50% of the maximum response for each cytokine concerning the inhibition of HPV-I6 LCR activity (data not shown) . All the combinations showed an increase of the inhibitory activity compared to the values obtained with the cytokines used alone, which is consistent with an additive effect (Table 2) .

Table 2. Effect of cytokines combinations on HPV-16LCR activity

% of LCR inhibition Cytokines (mean values + SD)

IL-4 (5 ng/ml) 37.3 ± 2.3

TGF-βl (5 ng/ml) 45.7 ± 3.8

IFN-α (100 U/ml) 53 ± 6.4

IFN-γ (100 U/ml) 21.5 ± 1.1

IL-4 (5 ng/ml) + TGF-βl (5 62.8 + 4.5 ng/ml)

IL-4 (5 ng/ml) + INF-α (100 76 + 6.5

U/ml)

IL-4 (5 ng/ml) + INF-γ (100 63.2 + 4.3

U/ml)

In order to determine whether the reduction in luciferase activity IL-4 and IL-13 exerted on the reporter clone P13 reflected a down-regulation of E6 and E7 mRNA levels in HPV- 16 positive cervical carcinoma cells, the CaSki cell line was selected as a model. First, the expression of the IL-4 and IL-13 receptor components, IL-4Rα-chain and IL-13Rαl-chain, in HaCat, CaSki and in a THP-I monocyte cell line (used as a positive control) was examined. Cytofluorimetric analysis revealed that HaCat and CaSki cells express both chains of the IL-4 and IL-13 receptor and are therefore potentially responsive to these cytokines (data not shown) . Next the effect of IL-4 and IL-13 on the expression of E6 and E7 genes in the CaSki cell line was studied. Since TGF-βl is a well- known inhibitor of HPV-I6 LCR activity and its receptor is expressed on CaSki cells, this cytokine was also included as a reference in this set of experiments. To this aim, the cells were treated with 50 ng/ml of the cytokines for 24 hours, and E6 and E7 mRNA levels were determined by Real-Time RT-PCR. The results demonstrate that all the cytokines reduced the E6 and E7 mRNA levels but to a different extent. The results are shown in Fig. 2. Specifically, Fig. 2 shows the effect of TGF-βl, IL-4 and IL-13 on the steady-state levels of E6 and E7 mRNAs in CaSki cells. CaSki cells were treated with 50 ng/ml of each cytokine for 24 hours or left untreated. mRNA was retrotranscribed, and the levels of HPV- 16 E6 and E7 transcripts were determined by Real-Time PCR. The results are shown as the mean of three independent experiments .

TGF-βl treatment resulted in a 86.2% inhibition of the E6 transcript and in a 85.5% inhibition of the E7 transcript. IL-4 treatment resulted in a 56.3% inhibition of the E6

transcript and in a 62% inhibition of the E7 transcript, while IL-13 treatment resulted in a 50% inhibition of the E6 transcript and in a 53% inhibition of the E7 transcript. These findings confirm the inhibitory activity on LCR-driven transcription observed with the reporter cell line after TGF- β, IL-4 and IL-13 treatment, and rules out the possibility that the decrease in luciferase activity might have been a consequence of an unspecific effect the cytokines exerted on enzyme activity or stability.

Discussion

The screening confirmed the inhibitory activity of. IFNs, TNF-α, and TGF-βl on the HPV-16 LCR. These results demonstrated the reliability of the assay.

The TGF-β superfamily includes TGF-β isoforms (TGF-βl, TGF- β2, TGF-β3) , activins, inhibins, growth and differentiation factors (GDFs) and bone morphogenetic proteins (BMPs) . These molecules act through specific receptor complexes composed of type I and type II serine/threonine receptor kinases. The receptor kinases subsequently activate Smad proteins which then propagate the signals into the nucleus to regulate target gene expression. The assay showed that besides TGF-βl, also TGF-β2 and TGF-β3 inhibit LCR activity, whereas a moderate or no effect was seen with Activin or GDF-15, respectively. Activin and TGF-β have been shown to differently regulate certain HaCaT differentiation markers, suggesting that TGF-β and activin may transduce, at least in part, different signals inside the cells. This may explain the different activity distinct members of the TGF-β superfamily have on the HPV LCR.

A novel finding of our study is the strong inhibitory activity IL-4 and IL-13 exerted on the LCR-driven transcription and their ability to down-regulate E6 and E7 mRNA levels in a HPV-I6 positive cervical carcinoma-derived cell line. IL-4 and IL-13 are structurally and functionally related and are secreted mainly by activated Th2 lymphocytes. Both cytokines exert a variety of effects on different cell types, including monocytes, B-lymphocytes, mast cells, and keratinocytes. IL-4 and IL-13 promote IgE isotype switching, enhance the expression of MHC class II on B cells, and down- regulate proinflammatory cytokine expression by monocytes. They also have proinflammatory effects on other cell types. In particular, they enhance IL-6 expression by endothelial cells and keratinocytes. These overlapping activities are conferred by the existence of common receptor components . The IL-4 receptor alpha-chain (IL-4Ra) and the IL-13 receptor alpha chain (IL-13Ra) can heterodimerize and form a receptor that binds either IL-4 or IL-13.

So far, IL-4 and IL-13 have not been considered antiviral cytokines, however, they have been shown to suppress HIV expression in monocytic cells and human macrophages. In addition, IL-4 has also been found to suppress the expression and replication of hepatitis B virus at the transcriptional level in the hepatocellular carcinoma cell line. These observations, along with the results presented here, point to an emerging role of IL-4 and IL-13 in host defence against viral infection, although their clinical significance remains to be explored.

In conclusion, the assay of the present invention provides a validated tool in the search for compounds that inhibit E6 and E7 gene transcription. Given its amenability to a high-

throughput format, the cell-based assay of the invention could greatly facilitate the discovery and development of drugs against HPV-associated human cancers. Moreover, the assay allowed the first systematic analysis of the effect exerted by cytokines on HPV-I6 LCR transcriptional activity.