Premium
A cell‐based high‐throughput assay for screening inhibitors of human papillomavirus‐16 long control region activity
Author(s) -
Lembo David,
Donalisio Manuela,
De Andrea Marco,
Cornaglia Maura,
Scutera Sara,
Musso Tiziana,
Landolfo Santo
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.05-3904fje
Subject(s) - transfection , reporter gene , drug discovery , biology , human papillomavirus , cancer research , high throughput screening , phenotypic screening , cell culture , cervical cancer , transcription (linguistics) , gene , computational biology , gene expression , phenotype , cancer , bioinformatics , medicine , genetics , linguistics , philosophy
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‐β1, TGF‐β2, TGF‐β3, TNF‐α, IFN‐α, and IFN‐β 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.