Modified DNA Fragments Specifically and Irreversibly Bind Transcription Factor NF-kappaB in Lysates of Human Tumor Cells

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M. A. Timchenko^1*, E. Yu. Rybalkina², A. Yu. Lomakin³, K. I. Evlakov³, I. N. Serdyuk³, and M. G. Ivanovskaya¹

¹Belozersky Institute of Physico-Chemical Biology and Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: img@genebee.msu.su; mtimchenko@vega.protres.ru

²Institute of Carcinogenesis, Blokhin Cancer Research Center, Russian Academy of Medical Sciences, 115478 Moscow, Russia; fax: (495) 324-1205; E-mail: rybalkina@crc.umos.ru

³Institute of Protein Research, 142290 Pushchino, Moscow Region, Russia; fax: (495) 924-0493; E-mail: serdyuk@vega.protres.ru

^* To whom correspondence should be addressed.

Received June 15, 2005; Revision received September 15, 2005

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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.

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KEY WORDS: transcription factor NF-kappaB, modified DNA duplexes, covalent binding, DNA-protein interactions

DOI: 10.1134/S0006297906040158

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