In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter.
Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.
Cervoni
Laura
L
Istituto Pasteur Fondazione Cenci Bolognetti, Università di Roma 'La Sapienza' P.le Aldo Moro 5, 00185 Roma, Italy.
Pietrangeli
Paola
P
Chichiarelli
Silvia
S
Altieri
Fabio
F
Egistelli
Lorenza
L
Turano
Carlo
C
Lascu
Ioan
I
Giartosio
Anna
A
D015153:Blotting, Western; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D020014:K562 Cells; D020559:Monomeric GTP-Binding Proteins / Q000187:drug effects* / Q000276:immunology; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D014157:Transcription Factors / Q000187:drug effects* / Q000276:immunology
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D054778:NM23 Nucleoside Diphosphate Kinases; D010982:Platelet-Derived Growth Factor; D014157:Transcription Factors; C075278:platelet-derived growth factor A; D005557:Formaldehyde; D004247:DNA; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins; D002945:Cisplatin