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Modified DNA fragments specifically and irreversibly bind transcription factor NF-kappaB in lysates of human tumor
M A Timchenko1, E Yu Rybalkina, A Yu Lomakin
1Belozersky Institute of Physico-Chemical Biology and Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia. mtimchenko@vega.protres.ru
Abstract:
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.
Insights
Synthetic DNA selectively binds to the NF-kappaB transcription factor in human colon cancer cells. This modified DNA can enter cells and partially translocate to the nucleus upon TNF-alpha stimulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear Factor kappa B (NF-kappaB) is a crucial transcription factor involved in cellular responses.
- Dysregulation of NF-kappaB is implicated in various cancers, including colon carcinoma.
- Targeting NF-kappaB offers a potential therapeutic strategy for cancer treatment.
Purpose of the Study:
- To investigate the covalent binding of a synthetic DNA fragment to NF-kappaB in human colon carcinoma cells.
- To assess the cellular uptake and intracellular localization of a modified DNA duplex.
- To explore the effect of TNF-alpha on the nuclear translocation of the DNA reagent.
Main Methods:
- Utilized 32P-labeled synthetic DNA duplex with a modified pyrophosphate group targeting the NF-kappaB recognition site.
- Employed denaturing gel electrophoresis (Laemmli) and immunoblotting for detecting DNA-NF-kappaB binding in cell lysates.
- Used rhodamine-labeled DNA for cellular delivery studies via fluorescent and confocal laser scanning microscopy.
Main Results:
- Demonstrated selective and irreversible covalent binding of the 32P-labeled DNA duplex to NF-kappaB in HCT-116 cell lysates.
- Observed that rhodamine-labeled DNA penetrates the plasma membrane of tumor cells without external delivery systems.
- Found initial cytoplasmic localization of the DNA, with partial nuclear translocation upon TNF-alpha treatment.
Conclusions:
- The synthetic DNA fragment effectively binds covalently to NF-kappaB in a cellular context.
- The modified DNA exhibits cell-penetrating properties and can be trafficked within the cell.
- TNF-alpha stimulation influences the intracellular distribution, promoting partial nuclear entry of the DNA reagent.
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