Modified DNA fragments specifically and irreversibly bind transcription factor NF-kappaB in lysates of human tumor cells.
Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-kappaB has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used 32P-labeled 17/19 bp nucleotide DNA duplex containing an NF-kappaB recognition site (kappaB-site) in which one of internucleotide phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 32P-labeled DNA duplex with NF-kappaB in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3 -end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-alpha promoted partial translocation of the DNA reagent into the nucleus.
Timchenko
M A
MA
Belozersky Institute of Physico-Chemical Biology and Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia. mtimchenko@vega.protres.ru
Rybalkina
E Yu
EY
Lomakin
A Yu
AY
Evlakov
K I
KI
Serdyuk
I N
IN
Ivanovskaya
M G
MG
D000906:Antibodies / Q000378:metabolism; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D045325:HCT116 Cells; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism / Q000494:pharmacology
D000906:Antibodies; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D016328:NF-kappa B; C468119:NF-kappaB decoy; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA