COMPLEMENTATION OF TRANSFORMED FIBROBLASTS FROM PATIENTS WITH COMBINED XERODERMA PIGMENTOSUM-COCKAYNE SYNDROME

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human heredi tary 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 bee n 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 cult ures from two patients, displaying characteristics associated with CS and belonging to XP complementation group Gr, were transformed with an origin-of-replication-deficient simian virus 40 (SV40). The new cell lines, Sigma XPCS1LVo- and Sigma XPCS1ROo-, were characterized phenoty pically and genotypically to verify that properties of the primary cel ls are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV 40 T antigen. The Sigma XPCS1LVo- and Sigma 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) am plification and restriction enzyme cleavage, the Sigma XPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was ev ident in the XPG RNA. Finally, to characterize the XPG DNA repair defi ciency in these cell lines, an episomal expression vector containing w ild-type XPG cDNA was used to correct UV-induced damage in a beta-gala ctosidase reporter gene. (C) 1998 Academic Press.