Abstract
The uppermost layer of our skin, the epidermis, is formed largely of keratinocytes which constitute the skin’s major barrier function and the first line of defence against environmental physical, chemical and biological agents. The subsequent layer, the dermis, which is mainly formed by fibroblasts, has a more supportive function, containing large amounts of collagen, blood vessels and nerve endings and is less directly affected by external insults. Hence it is likely that keratinocytes and fibroblasts have evolved different strategies to cope with the dangers of the environment. Mouse models with various genetic backgrounds in genome care-taking systems, such as DNA repair processes, are well suited to study differences between these two cell types and their implications for cancer and aging. In this chapter we describe a simple procedure to establish long-term keratinocyte and fibroblast cultures from, respectively, the epidermis and dermis of normal or NER-deficient newborn mice. The importance of the external O2 pressure during the establishment and maintenance of these matched cultures is discussed.
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References
Friedberg, E.C. (2001) How nucleotide excision repair protects against cancer. Nat Rev Cancer 1, 22–33.
Lehmann, A.R. (2003) DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101–11.
D'Errico, M., Lemma, T., Calcagnile, A., Proietti, D.S., Dogliotti, E. (2007) Cell type and DNA damage specific response of human skin cells to environmental agents. Mutat Res 614, 37–47.
van Oosten, M., Rebel, H., Friedberg, E.C., van Steeg, H., van der Horst, G.T., van Kranen, H.J., Westerman, A., van Zeeland, A.A., Mullenders, L.H., de Gruijl, F.R. (2000) Differential role of transcription-coupled repair in UVB-induced G2 arrest and apoptosis in mouse epidermis. Proc Natl Acad Sci USA 9, 11268–73.
Alekseev, S., Kool, H., Rebel, H., Fousteri, M., Moser, J., Backendorf, C., de Gruijl, F.R., Vrieling, H., Mullenders, L.H. (2005) Enhanced DDB2 expression protects mice from carcinogenic effects of chronic UV-B irradiation. Cancer Res 65, 10298–306.
Pines, A., Backendorf, C., Alekseev, S., Jansen, J.G., de Gruijl, F.R., Vrieling, H., Mullenders, L.H. (2008) Differential repair of UV photoproducts in murine keratinocytes and fibroblasts is mediated by differential activity of UV-DDB. DNA Repair (Amst) 8 N 53–61.
Backendorf, C., de Wit, J., van Oosten, M., Stout, G.J., Mitchell, J.R., Borgstein, A.M., van der Horst, G.T., de Gruijl, F.R., Brouwer, J., Mullenders, L.H., Hoeijmakers, J.H. (2005). Repair characteristics and differentiation propensity of long-term cultures of epidermal keratinocytes derived from normal and NER-deficient mice. DNA Repair (Amst) 4, 1325–36.
Stout, G.J., van Oosten, M., Acherrat, F.Z., de Wit, J., Vermeij, W.P., Mullenders, L.H., Gruijl, F.R., Backendorf, C. (2005) Selective DNA damage responses in murine Xpa-/-, Xpc-/- and Csb-/- keratinocyte cultures. DNA Repair (Amst) 4, 1337–44.
Stout, G.J., Westdijk, D., Calkhoven, D.M., Pijper, O., Backendorf, C., Willemze, R., Mullenders, L.H., de Gruijl, F.R. (2005). Epidermal transit of replication-arrested, undifferentiated keratinocytes in UV-exposed XPC mice: an alternative to in situ apoptosis. Proc Natl Acad Sci USA 27, 18980–85.
Rheinwald, J.G., Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–43.
Hager, B., Bickenbach, J.R., Fleckman, P. (1999) Long-term culture of murine epidermal keratinocytes. J Invest Dermatol 112, 971–76.
Caldelari, R., Suter, M.M., Baumann, D., de Bruin, A., Muller, E. (2000) Long-term culture of murine epidermal keratinocytes. J Invest Dermatol 114, 1064–65.
Menon, G.K., Grayson, S., Elias, P.M. (1985) Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. J Invest Dermatol 84, 508–12.
Lichti, U., Anders, J., Yuspa, S.H. (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3, 799–810.
Stücker, M., Struk, A., Altmeyer, P., Herde, M., Baumgärtl, H., Lübbers, D.W. (2002) The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis. J Physiol 538, 985–94.
Ngo, M.A., Sinitsyna, N.N., Qin, Q., Rice, R.H. (2007). Oxygen-dependent differentiation of human keratinocytes. J Invest Dermatol 127, 354–61.
Brennan, J.K., Mansky, J., Roberts, G., Lichtman, M.A. (1975) Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro. In Vitro 11, 354–60.
Werner, S. (1998) Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev 9, 153–65.
Steiling, H., Werner, S. (2003) Fibroblast growth factors: key players in epithelial morphogenesis, repair and cytoprotection. Curr Opin Biotechnol 14, 533–37.
Acknowledgements
CB would like to thank Ulrike Lichti, Dave Morgan, Adam Glick and Stuart Yuspa (NIH/NCI/CCR, Bethesda, MD) for their hospitality and help during early stages of this project. Jan Hoeijmakers (Genetics, EUR, Rotterdam), Leon Mullenders (Toxicology, LUMC, Leiden) and Frank de Gruijl (Dermatology, LUMC, Leiden) are acknowledged for supplying normal and NER-deficient mice and for their continuous interest and support.
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Pines, A., Backendorf, C. (2010). Matched Cultures of Keratinocytes and Fibroblasts Derived from Normal and NER-Deficient Mouse Models. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 585. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-380-0_4
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DOI: https://doi.org/10.1007/978-1-60761-380-0_4
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