Complementation of Transformed Fibroblasts from Patients with Combined Xeroderma Pigmentosum–Cockayne Syndrome

• Published in: Experimental cell research 1998-08, Vol.243 (1), p.22-28
• Main Authors: Ellison, Aaron R., Nouspikel, Thierry, Jaspers, Nicolaas G.J., Clarkson, Stuart G., Gruenert, Dieter C.
• Format: Article
• Language: English
• Subjects: Cell Line, Transformed - metabolism; Cell Line, Transformed - radiation effects; Cell Transformation, Viral; Cockayne Syndrome - complications; Cockayne Syndrome - genetics; DNA Repair - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Endonucleases; Fibroblasts - metabolism; Fibroblasts - radiation effects; Genotype; Humans; Nuclear Proteins; Phenotype; Simian virus 40; Transcription Factors; Ultraviolet Rays; Xeroderma Pigmentosum - complications; Xeroderma Pigmentosum - genetics
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1998.4147

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40).The new cell lines, ∑XPCS1LVo- and ∑XPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The ∑XPCS1LVo- and ∑XPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene.Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the ∑XPCS1ROo- cells were shown to retain the homozygous T deletion atXPGposition 2972. This mutation also characterizes the parental primary cells and was evident in theXPGRNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-typeXPGcDNA was used to correct UV-induced damage in a β-galactosidase reporter gene.