title: Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.
abstract: Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.
authors.affiliation: Department of Pharmacology & Toxicology, University of Louisville, 570 South Preston Street, Rm221, Louisville, KY 40202, USA.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Porter
authors.forename: Denise R
authors.initials: DR
authors.lastname: Clark
authors.forename: Lisa D
authors.initials: LD
authors.lastname: McDaniel
authors.forename: W Glenn
authors.initials: WG
authors.lastname: McGregor
authors.forename: J Christopher
authors.initials: JC
authors.lastname: States
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000528:radiation effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D005347:Fibroblasts / Q000502:physiology / Q000528:radiation effects; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000528:radiation effects; D008297:Male; D016296:Mutagenesis / Q000528:radiation effects*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays / Q000009:adverse effects*
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D012694:Serine; D013762:Tetrahydrofolate Dehydrogenase; D007041:Hypoxanthine Phosphoribosyltransferase; D019098:Telomerase