title: HPV-DNA is not detectable in outgrowing cells from explant cultures of skin lesions established at the air-liquid-interface.
abstract: Keratinocyte cultures established from HPV containing skin cancers were described earlier to lose their HPV DNA after passaging in vitro. A different approach was therefore used in this study. Explant cultures were generated by depositing small pieces of various benign and (pre)malignant skin specimens of renal transplant recipients and non-immunosuppressed patients on fibroblast-populated collagen lattices or on de-epidermized dermis. Subsequently, the cultures were maintained at the air-liquid interface. At various time points, samples were collected for both HPV analysis, using a nested PCR approach, and morphology. The outgrowing keratinocytes developed into multilayered epithelial structures showing terminal differentiation. No histological differences were observed between cultures established from HPV positive and negative lesions. Eighteen biopsy specimens were tested for their HPV content before and after culture. Before culture 11 out of these skin specimens contained DNA of the Epidermodysplasia Verruciformis-related HPV types (EV-HPV). Comparison of the HPV types detected in two different parts of the same skin specimen before culture was strongly suggestive for a non-homogeneous distribution of EV-HPV in the lesions. From the explant cultures derived from the 11 HPV-positive biopsies, 31 samples from the originally explanted pieces of tissue and 38 samples from the outgrowing multilayered epithelial sections were collected. HPV DNA was detected in 10 of the 31 and in 3 of the 38 samples (Chi-square test, P = 0.01), respectively. These results indicate that EV-HPV positive keratinocytes do not efficiently proliferate or lose their HPV DNA in this culture system or EV-HPV DNA is present in only a few basal cells, making it improbable that these cells are located at the outgrowing margins.
authors.affiliation: Department of Virology, Academic Medical Centre, Amsterdam, The Netherlands.
authors.forename: I L
authors.initials: IL
authors.lastname: Boxman
authors.forename: L H
authors.initials: LH
authors.lastname: Mulder
authors.forename: B J
authors.initials: BJ
authors.lastname: Vermeer
authors.forename: J N
authors.initials: JN
authors.lastname: Bavinck
authors.forename: J
authors.initials: J
authors.lastname: ter Schegget
authors.forename: M
authors.initials: M
authors.lastname: Ponec
mesh_terms: D000328:Adult; D000368:Aged; D046508:Culture Techniques / Q000379:methods; D004279:DNA, Viral / Q000032:analysis*; D004819:Epidermodysplasia Verruciformis / Q000821:virology*; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000821:virology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012878:Skin Neoplasms / Q000821:virology*; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000821:virology
chemical_list: D004279:DNA, Viral