Xeroderma pigmentosum variant: generation and characterization of fibroblastic cell lines transformed with SV40 large...

Xeroderma pigmentosum variant (XPV) fibroblasts from the XP4BE strain (CRL1162) were transformed with the SV40 large T antigen with the purpose of generating immortalized cell lines that are defective in post-replication repair (PRR). Two transformation and selection protocols were used and at least two independent clones were obtained, which behaved in culture as immortal cell lines. Fingerprinting analyses were used to demonstrate their origin from XP4BE cells and to compare their genetic profiles. These cell lines were shown to be hypersensitive to killing by uv when compared to SV40-transformed fibroblasts derived from foreskins of normal neonates. One of the XPV transformed cell lines (CTag) was characterized further as a potential source of cell-free extracts with capability for catalyzing the T antigen-dependent in vitro replication of plasmid DNA carrying the SV40 origin of replication. In this assay system, CTag extracts were shown to be as active as those produced from HeLa cells. In vitro replication of uv-damaged plasmid DNA by protein extracts from PRR-defective (XPV) and PRR-proficient cells might allow the identification and characterization of protein factors that contribute to normal replication of uv-damaged DNA. Ultraviolet irradiation of plasmid DNA templates caused dose-dependent inhibition of in vitro replication by both HeLa and CTag extracts.