| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 27, 2005 as doi:10.1096/fj.05-3904fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Public Health and Microbiology, University of Turin, Italy
1Correspondence: Via Santena 9, Turin 10126, Italy. E-mail: david.lembo{at}unito.it
SPECIFIC AIMS
The continuous expression of human papillomavirus (HPV) E6 and E7 oncoproteins in cervical cancers is necessary for maintenance of the transformed phenotype and so represents a target for antiviral therapy. To provide a tool for the discovery and development of drugs against HPV-associated cancers, we devised a cell-based high-throughput (HT) screening system that allows easy, fast and reliable detection of inhibitors of HPV-16 E6 and E7 oncoprotein expression in libraries of synthetic or biological compounds.
PRINCIPAL FINDINGS
1. Generation of reporter cell clones expressing luciferase under the control of the enhancer-promoter region of the E6 and E7 genes (LCR)
The HaCaT human keratinocyte cell line was co-transfected with the reporter plasmid pALuc HPV-16-LCR, which contains the complete LCR fragment of the HPV-16 cloned in front of the firefly luciferase gene, and with pSTneoB, which confers high G418 resistance to mammalian cells. After antibiotic selection and cloning by limiting dilution, two clones designated P13 and P21, which showed high luciferase activity, were chosen for further characterization.
2. Characterization and validation of reporter clones P13 and P21
Standard PCR for the LCR confirmed the presence of an integrated reporter construct (Fig. 1
A). The LCR copy number is 1 copy/genome for both clones as estimated by quantitative real-time (RT) PCR. The reporter clones were then validated for use in a cell-based assay by measuring 1) stability of luciferase expression over time; 2) response to known inhibitory stimuli; 3) compatibility with a HT format. Stable levels of luciferase activity were detected in P13 and P21 clones collected on days 30, 40, 50, 60, 70, and 80 post-selection (data not shown). A 24 h treatment of the reporter clones with the two known LCR activity inhibitors, cytokines TGF-ß1 and TNF-
, significantly decreased luciferase activity (Fig. 1B
). To increase assay throughput, we optimized the test conditions for use in a 96-well plate format with respect to the cell number per well. An easily detectable signal was obtained by plating 20,000 cells per well (Fig. 1C
). Taken together, these results demonstrate that the reporting clones could be used for screening inhibitors of LCR activity in a HT fashion.
|
3. Screening of a panel of cytokines using the HPV-16 LCR reporter cell line
To exploit the cell-based assay, we used clone P13 to screen a panel of 32 cytokines belonging to different functional classes in a 24-well plate format. A diagram of the screening scheme is presented in Fig. 2
. 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-1ß, and IFN-
, that exerted moderate inhibitory activity (30–49%); group III comprised the eight cytokines—IL-4, IL-13, TGF-ß1, TGF-ß2, TGF-ß3, TNF-, IFN-, and IFN-ß—that exerted high inhibitory activity (50–70%). None showed antiproliferative activity at the concentration and time point chosen for the luciferase assay, indicating that reduction in luciferase activity is not a consequence of reduced cell proliferation or viability. IL-4 and IL-13 had never been reported as inhibitors of LCR transcriptional activity; therefore, they were selected for a more detailed examination. The inhibitory dose producing a 50% inhibition of LCR activity (ID50) is 4 ng/mL for IL-4 and 11.3 ng/mL for IL-13.
|
|
4. Effect of IL-4 and IL-13 on HPV-16 E6 and E7 mRNA levels in the CaSki cell line
Since the LCR drives the transcription of the E6 and E7 genes in the HPV genome, we used RT-PCR to determine whether the reduction in luciferase activity induced by IL-4 and IL-13 on the reporter clone reflected a down-regulation of E6 and E7 mRNA levels in the HPV-16 positive cervical carcinoma CaSki cell line. 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. This finding confirms the inhibitory activity of IL-4 and IL-13 on LCR-driven transcription and rules out the possibility that the decrease in luciferase activity might have been a consequence of an unspecific effect of the cytokines on enzyme activity or stability.
CONCLUSIONS AND SIGNIFICANCE
Cervical cancer is the second most prevalent cancer and ranks fifth as the cause of cancer deaths in women. Current treatment for HPV-associated tumors is surgery. However, even after surgical removal of the neoplasm, tumor growth may recur due to persistence of the virus in healthy tissues. Prevention of HPV infection by vaccination or immunotherapy is under investigation but not yet established. Antiviral strategies are therefore needed to treat HPV-induced neoplasia and to reduce its recurrence. Although the major mechanisms in HPV-induced transformation have been elucidated, the race for drugs against HPV-associated cervical cancer has been hampered by two major obstacles: 1) systems to study HPV in vitro are technically difficult and not convenient for screening anti-viral compounds at an early stage of development; 2) the HPV genome does not encode enzyme activities such as polymerases or proteases, which are traditional targets for antiviral therapies.
HPV are causative agents of virtually all cases of cervical carcinomas, and the continuous expression of the viral E6 and E7 oncoproteins is necessary for maintenance of the malignant phenotype. The E6 and E7 oncoproteins subvert cellular functions such as cell-cycle control, apoptosis, senescence and DNA repair by inducing degradation of tumor suppressor proteins p53 and p105Rb. Cervical carcinomas represent a peculiar target for antitumor therapy. Unlike many other tumor types, they frequently retain wild-type p53 and p105Rb and so are potentially susceptible to therapeutic strategies aimed at restoring growth control by repressing HPV oncoprotein expression. To date, no antiviral drug is available, so the only treatment for cervical cancers is surgery. Our assay provides a validated tool in the search for E6 and E7 transcription inhibitors in libraries of natural or synthetic compounds. Given its amenability to the HT format, our cell-based assay could greatly facilitate the discovery and development of drugs against HPV-associated human cancers. Moreover, with this assay the effect of cytokines on HPV-16 LCR transcriptional activity was systematically analyzed for the first time. These data may furnish information for guiding future studies of the role cytokines play in the clearance of the virus from infected tissues and in the control of persistent HPV infections. Furthermore, the discovery that IL-4 and IL-13 inhibit E6 and E7 expression warrants investigation into determining whether treatment of cervical carcinoma cells with these cytokines restores the p53 and p105Rb pathways and growth control.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3904fje;
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
