Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells.
We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.
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Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Moses
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D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects; D005199:Fanconi Anemia / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects* / Q000528:radiation effects; D006801:Humans; D008730:Methoxsalen; D010957:Plasmids / Q000187:drug effects* / Q000528:radiation effects; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D003432:Cross-Linking Reagents; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D008730:Methoxsalen