A cell‐based high‐throughput assay for screening inhibitors of human papillomavirus‐16 long control region activity

Abstract

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.

Interferon-Gamma Enhances TLR3 Expression and Anti-Viral Activity in Keratinocytes

“…The results of the present study showed that IFN-γ induces TLR3 expression and enhances anti-viral activity in NHEKs. IFN-γ alone, IFN-β, and some antimicrobial peptides have also anti-viral activities (Hazrati et al, 2006;Lembo et al, 2006;Crack et al, 2012;Wilson et al, 2013). Among them, IFN-β is reported to be the major factor responsible for anti-viral activity in human keratinocytes (Torseth et al, 1987;Fujisawa et al, 1997).…”

The Interplay between Antiviral Signalling and Carcinogenesis in Human Papillomavirus Infections

“…High-risk Intraepithelial neoplasia and cervical cancer 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 68, 73, 82 (26, 53, 66) Low-risk Intraepithelial neoplasia or genital warts 6,11,40,12,43,44,53,54,61,72,73,81 HPV particles are composed by a non-enveloped icosahedral capsid that shields the viral genome. The HPV genome consists of a single circular double-stranded DNA (dsDNA) molecule associated with host-derived histones, and, typically, encodes seven to eight open reading frames (ORFs) (Figure 1).…”

“…It was reported that several cytokines, namely IL-1α, IL-4 IL-13, transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α), IFN-α and IFN-β, act as inhibitors of HPV16 URR activity, suppressing early gene transcription [54]. Furthermore, type I IFNs, TNF-α and TGF-β restrain the growth of non-infected and HPV-infected keratinocytes, while this suppression tends to cease in oncogenic cells [55,56].…”

Early Defensive Mechanisms against Human Papillomavirus Infection

“…KC constitutively secrete low levels of soluble immune mediators, including cytokines, chemokines, and growth factors (46)(47)(48), the production of which can be elevated in response to proinflammatory stimuli, including PRR activation and autocrine or paracrine stimulation with inflammatory cytokines (31,43,46,47,49). Several cytokines, including type I IFNs, TNF-␣, IL-1␣, IL-4, IL-13, and transforming growth factor ␤ (TGF-␤), share the ability to inhibit HPV early gene expression (50)(51)(52)(53)(54). In addition, type I IFNs, TNF-␣, and TGF-␤ suppress the growth of normal KC and nontumorigenic, HPV-transformed KC, although effectiveness can vary with the transforming HPV type and is generally lost with progression to a tumorigenic phenotype (50,51,54,55).…”