Suboptimal action of NF-kappaB in Fanconi anemia cells results from low levels of thioredoxin.
Electrophoretic mobility shift assays (EMSA) revealed that under standard cell culture conditions NF-kappaB was induced in Fanconi anemia fibroblasts in contrast to control cells. Dithiothreitol, a potent synthetic redox potential-delivering compound, when added to growing cells, prevented this induction of NF-kappaB and, simultaneously, chromosomal instability was reduced. Fanconi anemia cells possess low endogenous levels of the naturally occurring antioxidant thioredoxin. Transfection of Fanconi anemia cells with thioredoxin cDNA containing a nuclear localization signal prevented both spontaneous as well as mitomycin C-induced chromosomal instability. A promotor construct with two NF-kappaB binding sites in front of the CAT gene induced little CAT expression in cells with low thioredoxin content in spite of induced NF-kappaB. In cells with higher thioredoxin content CAT expression was increased. Cotransfection of the NF-kappaB-dependent CAT plasmid with the Trx/nuc-plasmid into FA fibroblasts increased the CAT expression to almost that of control cells, indicating that in this model system with diminished thioredoxin content NF-kappaB requires thioredoxin for binding to its specific promotor. Since Fanconi anemia cells have low thioredoxin contents, NF-kappaB-dependent genes are expressed insufficiently. This explains part of the pathophysiological processes observed in Fanconi anemia.
Kontou
Maria
M
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Adelfalk
Caroline
C
Hirsch-Kauffmann
Monica
M
Schweiger
Manfred
M
D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D043171:Chromosomal Instability / Q000187:drug effects; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D005199:Fanconi Anemia / Q000188:drug therapy / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013879:Thioredoxins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D016328:NF-kappa B; D013879:Thioredoxins; D015500:Chloramphenicol O-Acetyltransferase; D004229:Dithiothreitol