A cell-based high-throughput assay for screening inhibitors of human papillomavirus-16 long control region activity.
Cervical carcinomas express human papillomavirus (HPV) E6 and E7 oncoproteins, which are required to maintain the proliferative state of cancer cells. Repression of E6 and E7 expression results in acquisition of senescent phenotype and in the rescue of functional p53 and p105(Rb) pathways; therefore, therapies directed against either viral protein may be beneficial. However, the systems to study HPV in vitro are technically difficult and not convenient for screening of antiviral compounds. This has hampered the discovery of drugs against HPV. Here we describe the generation and use of a high-throughput screening system based on keratinocytes stably transfected with a reporter construct containing the regulatory sequence of E6 and E7 gene transcription (LCR) that allows easy detection of inhibitors of E6 and E7 expression in libraries of synthetic or biological compounds. The assay was used to screen a wide panel of cytokines: among them, IL-4, IL-13, TGF-beta1, TGF-beta2, TGF-beta3, TNF-alpha, IFN-alpha, and IFN-beta were found to induce a strong inhibition of the LCR activity. Our assay provides a validated tool in the search for drugs against HPV-associated cervical carcinomas and allowed the first systematic analysis of the effect of cytokines on the HPV-16 LCR transcriptional activity.
Lembo
David
D
Department of Public Health and Microbiology, University of Turin, Italy. david.lembo@unito.it
Donalisio
Manuela
M
De Andrea
Marco
M
Cornaglia
Maura
M
Scutera
Sara
S
Musso
Tiziana
T
Landolfo
Santo
S
D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D018793:Interleukin-13 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects* / Q000821:virology; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000187:drug effects*
C068651:E6 protein, Human papillomavirus type 16; D018793:Interleukin-13; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D015847:Interleukin-4