title: Immortalization of human urothelial cells by human papillomavirus type 16 E6 and E7 genes in a defined serum-free system.
abstract: Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract.
  
  
  OBJECTIVE
  Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum-free culture system.
  
  
  RESULTS
  Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7.
  
  
  CONCLUSIONS
  The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.
authors.affiliation: Program in Human Urothelial Biology, Seattle Children's Hospital, Seattle, WA 98105, USA.
authors.forename: N
authors.initials: N
authors.lastname: Carmean
authors.forename: J W
authors.initials: JW
authors.lastname: Kosman
authors.forename: E M
authors.initials: EM
authors.lastname: Leaf
authors.forename: A E
authors.initials: AE
authors.lastname: Hudson
authors.forename: K E
authors.initials: KE
authors.lastname: Opheim
authors.forename: J A
authors.initials: JA
authors.lastname: Bassuk
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005814:Genes, Viral; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D007621:Karyotyping; D007633:Keratins / Q000378:metabolism; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections; D010641:Phenotype; D011799:Quinazolines / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics*; D019098:Telomerase / Q000378:metabolism; D019459:Urothelium / Q000166:cytology* / Q000187:drug effects
chemical_list: D016895:Culture Media, Serum-Free; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011799:Quinazolines; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D004815:Epidermal Growth Factor; D007633:Keratins; D066246:ErbB Receptors; D019098:Telomerase; C088860:4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline