Using an Immortalized Cell Line to Study the HPV Life Cycle in Organotypic

Abstract

The papillomavirus life cycle is tied to the differentiation of the stratified squamous epithelium that this virus infects. The ability to study the papillomavirus life cycle is facilitated by organotypic culturing techniques that allow one to closely recapitulate this terminal differentiation process in the laboratory. Current techniques allow for the establishment of recombinant wild-type or mutant human papillomavirus (HPV) genomes in transfected early-passage human foreskin keratinocytes (HFKs). These cells can then be used in organotypic culture to investigate the role of individual viral genes in different aspects of the viral life cycle. When using early-passage HFKs, there is a need for the transfected HPV genome to extend the life span of the cells in order to have sufficient cell generations in which to carry out organotypic culturing. The recent isolation of a spontaneously immortalized HFK cell line that supports the complete HPV life cycle has further allowed investigators to study wild-type or mutant papillomaviral genomes that do not confer immortalization. In this chapter, we describe the methodologies that permit the study of the HPV life cycle in this HFK cell line.

Reconstruction of Human Papillomavirus Type 16-Mediated Early-Stage Neoplasia Implicates E6/E7 Deregulation and the Loss of Contact Inhibition in Neoplastic Progression

“…1C and E), with E4 expression being supported in only a few cells in the uppermost layers. The various raft phenotypes were reproducible in duplicate wells in each experiment and were apparent in three independent experiments using a protocol that allowed a gradual growth to confluence on dermal equivalent (for 4 to 5 days) prior to lifting to the air-liquid interface (12). Plating at higher cell densities followed by more immediate lifting resulted in similar but less pronounced phenotypic differences, suggesting that a gradual establishment of the confluent monolayer is an important part of the protocol.…”

“…To study episomal HPV gene expression in the context of the virus life cycle, linear 8-kb HPV-16 (W12) genomes were recircularized using a dilute ligation reaction and introduced into the isogenic keratinocyte cell line (NIKS) (1) by cotransfection with a blastocidin drug resistance plasmid, using Effectene transfection reagent. Following selection, colonies were expanded into individual HPV-16 clonal cell lines for raft culture experiments (12). The parental NIKS cell, which is immortal but not transformed, is wild type for p16, p53, and pRb and retains its ability to differentiate normally in organotypic raft culture, characteristics which have recently led to its clinical application in the production of temporary skin grafts.…”

“…Early and late viral gene expression was subsequently examined in the NIKS HPV16 clonal cell lines after propagation in raft culture, using suprabasal MCM-7 as a surrogate marker of E7 (2,15) and HPV-16 E4 as a virally encoded marker of cells supporting genome amplification and late viral gene expression (5,15,19). The utility of the MCM-7 biomarker was validated as an E7 surrogate using NIKS lines that express only E6 or E7 or E6 and E7 together (12,15,18). In these studies, MCM-7 expression extended above the basal layer only in raft cultures expressing E7 (either alone or with E6) (Fig.…”

Sumoylation dynamics during keratinocyte differentiation

“…Human foreskin keratinocytes were grown in organotypic raft cultures to form stratified epithelium as previously described (Lambert et al, 2005). All chemicals used for immunohistochemistry were from Biocare Medical (Concord, CA).…”

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

“…Since transfection of nonsupercoiled DNA is inefficient and PHKs senesce after only a few passages, selection and expansion of drug-resistant colonies depend on HR HPV oncogenes to immortalize PHKs. One alternative has been to use certain immortalized epithelial cell lines as recipient cells (Fang et al 2006b; for review, see Lambert et al 2005). In either case, only a small fraction of spinous cells in raft cultures of immortalized cells support viral DNA amplification and progeny virus production.…”