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WO2006045833A1 - Procede de dosage d'identification d'inhibiteurs de l'expression des oncoproteines e6 et e7 du papillomavirus humain (hpv), cellule hote recombinee et kit d'application de ce procede - Google Patents

Procede de dosage d'identification d'inhibiteurs de l'expression des oncoproteines e6 et e7 du papillomavirus humain (hpv), cellule hote recombinee et kit d'application de ce procede Download PDF

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WO2006045833A1
WO2006045833A1 PCT/EP2005/055598 EP2005055598W WO2006045833A1 WO 2006045833 A1 WO2006045833 A1 WO 2006045833A1 EP 2005055598 W EP2005055598 W EP 2005055598W WO 2006045833 A1 WO2006045833 A1 WO 2006045833A1
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hpv
host cell
activity
expression
proteins
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Santo Landolfo
David Lembo
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Universita degli Studi di Torino
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Universita degli Studi di Torino
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • 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.
  • HPV human papilloma virus
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the luciferase activity and the activity of the reporter gene of the normalization plasmid 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.
  • the normalized luciferase activity is in turn divided by the concentration of the proteins obtained by the Bradford method.
  • 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.) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the assay of the invention comprises the steps of:
  • 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.
  • 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.
  • 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.
  • a recombinant nucleic acid construct for example recombinant DNA
  • 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.
  • 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.
  • 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) .
  • CAT chloramphenicol acetyl transferase
  • beta galactosidase beta galactosidase
  • alkaline phosphatase alkaline phosphata
  • 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.
  • 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.
  • kits 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.
  • 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:
  • sample preparation operations such as for example the removal of the medium, washing, harvesting and centrifugation of the cells, and the protein estimation
  • sample preparation operations such as for example the removal of the medium, washing, harvesting and centrifugation of the cells, and the protein estimation
  • 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.
  • the luciferase activity can be simultaneously revealed in the 96 wells of the plate with a suitable luminometer, thus decreasing the analysis times.
  • 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 epithelial cell line of the invention about 20 ⁇ g 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.
  • the plasmid pST-Neo Kerath et al. , Cell Structure and Function 2: 575-580, 1987
  • the cells were cultured in a medium containing 400 ⁇ g/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.
  • 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 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/ ⁇ g of protein being detected, which remained constant with time.
  • the assay was subjected to a validation assay using two known inhibitory stimuli: the cytokines TGF- ⁇ l and TNF- ⁇ (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.
  • 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 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.
  • clone P13 Screening of a panel of cytokines using the HPV-16LCR reporter cell line (clone P13)
  • 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-l ⁇ and IFN- ⁇ , that exerted a moderate inhibitory activity (30-49%) ;
  • Group III comprised the 8 cytokines, namely IL-4, IL-I3, TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, TNF- ⁇ , IFN- ⁇ and IFN- ⁇ , that exerted a high inhibitory activity (50-70%) .
  • No stimulatory cytokine was detected by the assay.
  • 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.
  • ID 50 the dose of IL-4 and IL-13 which produces a 50% inhibition of LCR activity
  • 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.
  • Nonlinear regression data analysis calculated an ID 50 of 4 ng/ml for IL-4 and 11.3 ng/ml for IL-13.
  • IL-4 (5 ng/ml) + TGF- ⁇ l (5 62.8 + 4.5 ng/ml)
  • the CaSki cell line was selected as a model.
  • the expression of the IL-4 and IL-13 receptor components, IL-4R ⁇ -chain and IL-13R ⁇ l-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) .
  • FIG. 2 shows the effect of TGF- ⁇ l, 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- ⁇ l 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.
  • the screening confirmed the inhibitory activity of. IFNs, TNF- ⁇ , and TGF- ⁇ l on the HPV-16 LCR. These results demonstrated the reliability of the assay.
  • the TGF- ⁇ superfamily includes TGF- ⁇ isoforms (TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3) , 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- ⁇ l, also TGF- ⁇ 2 and TGF- ⁇ 3 inhibit LCR activity, whereas a moderate or no effect was seen with Activin or GDF-15, respectively.
  • TGF- ⁇ and activin have been shown to differently regulate certain HaCaT differentiation markers, suggesting that TGF- ⁇ and activin may transduce, at least in part, different signals inside the cells. This may explain the different activity distinct members of the TGF- ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une cellule hôte épithéliale ayant subi une transfection stable par une construction d'acide nucléique recombinée comprenant la séquence d'acide nucléique d'un gène rapporteur, par exemple la luciférase, liée de manière fonctionnelle à une seconde séquence d'acide nucléique comprenant les séquences activatrices ainsi que le promoteur des gènes codant les protéines E6 et E7 du HPV. La cellule hôte de l'invention est utilisée comme système rapporteur dans un dosage de cellules à haut rendement, destiné à identifier des molécules utilisées pour traiter les tumeurs provoquées par le papillomavirus. Par ailleurs, l'invention concerne un kit de réalisation de ce dosage.
PCT/EP2005/055598 2004-10-29 2005-10-27 Procede de dosage d'identification d'inhibiteurs de l'expression des oncoproteines e6 et e7 du papillomavirus humain (hpv), cellule hote recombinee et kit d'application de ce procede Ceased WO2006045833A1 (fr)

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ITTO2004A000752 2004-10-29
ITTO20040752 ITTO20040752A1 (it) 2004-10-29 2004-10-29 Procedimento di saggio per identificare inibitori dell'espressione delle oncoproteine e6 e e7 del papilloma virus umano ( hpv ) cellula ospite ricombinante e kit per l'uso del suddetto procedimento

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881636A (zh) * 2020-07-03 2022-01-04 沈剑萍 一种游离细胞标记及分离探针及其相关产品和用途

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WO1999021979A1 (fr) * 1997-10-28 1999-05-06 Maxygen, Inc. Vecteurs du papillomavirus humain
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881636A (zh) * 2020-07-03 2022-01-04 沈剑萍 一种游离细胞标记及分离探针及其相关产品和用途
CN113881636B (zh) * 2020-07-03 2024-05-03 贝勒知临(杭州)生物技术有限公司 一种游离细胞标记及分离探针及其相关产品和用途

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