Mechanisms of the carcinogenic chromium(VI)-induced DNA-protein cross-linking and their characterization in cultured intact human cells.
DNA-protein complexes (DPCs) were induced in human leukemic T-lymphocyte MOLT4 cells by treatment with potassium chromate. DPCs were isolated by ultracentrifugal sedimentation in the presence of 2% SDS and 5 M urea. The complexes were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis. Three acidic proteins of 74, 44, and 42 kDa and a basic protein of 51 kDa were primarily complexed to DNA following 25 microM chromate treatment. Higher concentrations of chromate cross-linked many other proteins to DNA. Amino acid sequencing and immunoblotting studies indicated that the acidic 44-kDa protein could be nuclear beta-actin. Lectin and aminoglycoside nucleotidyltransferase were also found to cross-link with DNA by chromate treatment. The composition and stability of the DPCs were studied using nucleases, proteinase K, and disruptive chemicals. Pretreatment of cells with antioxidants inhibited the formation of DPCs, measured as K+-SDS precipitable DPCs, indicating the involvement of oxidative mechanisms. Because chromate causes certain nuclear proteins to form complexes with DNA and the complexes are resistant to treatments such as 2% SDS and 5 M urea, but disruptable under gel electrophoretic conditions, chromium could be used as a cross-linking agent for the identification of other proteins, such as transcription factors, that transiently interact with DNA.
Mattagajasingh
S N
SN
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0442, USA. misra@vt.edu
Misra
H P
HP
D002857:Chromium / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D013347:Subcellular Fractions / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D002857:Chromium; D004247:DNA; D003851:Deoxyribonucleases