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, XpdTTD 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.

Additional Metadata
Keywords Cockayne syndrome, Mouse keratinocyte cell lines, Nucleotide excision repair, Terminal differentiation, Tetraploidy, Trichothiodystrophy, Xeroderma pigmentosum
Persistent URL dx.doi.org/10.1016/j.dnarep.2005.07.011, hdl.handle.net/1765/68246
Journal D N A Repair
Citation
Backendorf, C.M.P, de Wit, J, van Oosten, M, Stout, G.J, Mitchell, J.R, Borgstein, A.M, … Hoeijmakers, J.H.J. (2005). Repair characteristics and differentiation propensity of long-term cultures of epidermal keratinocytes derived from normal and NER-deficient mice. D N A Repair, 4(11), 1325–1336. doi:10.1016/j.dnarep.2005.07.011