title: Repair characteristics and differentiation propensity of long-term cultures of epidermal keratinocytes derived from normal and NER-deficient mice.
abstract: Epidermal keratinocytes constitute the most relevant cellular system in terms of DNA damage because of their continuous exposure to UV light and genotoxic chemicals from the environment. Here, we describe the establishment of long-term keratinocyte cultures from the skin of wild-type and nucleotide excision repair (NER) deficient mouse mutants. The use of media with a lowered calcium concentration and the inclusion of keratinocyte growth factor (KGF) permitted repeated passaging of the cultures and resulted in the generation of stable cell lines that proliferated efficiently. The cells retained their normal ability to engage into terminal differentiation when triggered with high calcium concentrations or after suspension in semi-solid medium. The cultures reflected the cellular characteristics (i.e. repair and transcription profiles) of the Xpa(-/-), Xpc(-/-), Csb(-/-) and Xpd(TTD) mouse models from which they were derived. For instance, in line with earlier in vivo results, Xpd(TTD) keratinocytes were disturbed in their ability to terminally differentiate in vitro. This was concluded from a delay in calcium-induced stratification and by reduced transcription of both early (keratin 10) and late (loricrin) terminal differentiation marker genes. UDS measurements in wild-type cells committed to terminal differentiation did not reveal any reduction in global DNA repair that could be indicative of differentiation associated repair (DAR) as found in neurons. UV sensitivity data revealed that in keratinocytes global genome repair contributes more to cell survival than previously concluded from fibroblast studies. It is inferred that these fully controllable in vitro cultures will be a valuable tool to assess critical parameters of genome care-taking systems in cell proliferation and differentiation.
authors.affiliation: Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands. backendo@chem.leidenuniv.nl
authors.forename: Claude
authors.initials: C
authors.lastname: Backendorf
authors.forename: Jan
authors.initials: J
authors.lastname: de Wit
authors.forename: Marijke
authors.initials: M
authors.lastname: van Oosten
authors.forename: Gerdine J
authors.initials: GJ
authors.lastname: Stout
authors.forename: James R
authors.initials: JR
authors.lastname: Mitchell
authors.forename: Anne-Marijke
authors.initials: AM
authors.lastname: Borgstein
authors.forename: Gijsbertus T
authors.initials: GT
authors.lastname: van der Horst
authors.forename: Frank R
authors.initials: FR
authors.lastname: de Gruijl
authors.forename: Jaap
authors.initials: J
authors.lastname: Brouwer
authors.forename: Leon H F
authors.initials: LH
authors.lastname: Mullenders
authors.forename: Jan H J
authors.initials: JH
authors.lastname: Hoeijmakers
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics* / Q000502:physiology; D045643:DNA Repair Enzymes / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004195:Disease Models, Animal; D000078404:Epidermal Cells; D004817:Epidermis / Q000473:pathology / Q000502:physiology; D015603:Keratinocytes / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D000075223:Poly-ADP-Ribose Binding Proteins; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics; D051759:Xeroderma Pigmentosum Group D Protein / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D051760:Xeroderma Pigmentosum Group A Protein; C501002:Xpc protein, mouse; C513075:Ercc6 protein, mouse; D051759:Xeroderma Pigmentosum Group D Protein; C497293:Ercc2 protein, mouse; D045643:DNA Repair Enzymes