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Double-Stranded DNA Fragments Bearing Unrepairable Lesions and Their Internalization into Mouse Krebs-2 Carcinoma
Evgeniya V Dolgova1, Alexey N Evdokimov2, Anastasia S Proskurina1
1Laboratory of Induced Cell Processes, Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Abstract:
Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 μL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 μg per 106 cells. For the similar unmodified DNA fragments, this ratio is 0.73 μg per 106 cells. Krebs-2 tumor cells were shown to be saturated internalizing either (190 ± 40) × 103 molecules of modified DNA or (1,000 ± 100) × 103 molecules of native DNA. On internalization, the fragments of DNA undergo partial and nonuniform hydrolysis of 3' ends followed by circularization. The degree of hydrolysis, assessed by sequencing of several clones with the insertion of specific PCR product, was 30-60 nucleotides.
Insights
Murine Krebs-2 tumor cells internalize extracellular DNA, which interferes with DNA repair. Modified DNA fragments were found to inhibit nucleotide excision repair, with specific internalization parameters characterized.
Area of Science:
- Molecular Biology
- Cancer Research
- DNA Repair Mechanisms
Background:
- Murine Krebs-2 tumor-initiating stem cells possess the ability to internalize extracellular double-stranded DNA fragments.
- Internalized DNA fragments can disrupt the repair of chemically induced interstrand cross-links within cells.
Purpose of the Study:
- To characterize the internalization of modified extracellular DNA by murine Krebs-2 tumor cells.
- To investigate the impact of modified DNA on DNA repair pathways, specifically nucleotide excision repair.
Main Methods:
- Utilized a 756 bp polymerase chain reaction (PCR) product containing a bulky photoreactive dC adduct as modified extracellular DNA.
- Characterized DNA probe internalization parameters including incubation conditions, percentage of cells internalizing DNA, and saturation levels.
- Assessed DNA hydrolysis and circularization post-internalization using sequencing of PCR product clones.
Main Results:
- Modified DNA inhibited nucleotide excision repair and was resistant to excision by this system.
- Under standard conditions, 0.35% ± 0.18% of Krebs-2 ascites cells internalized modified DNA.
- Saturation occurred at 0.37 μg/10^6 cells for modified DNA versus 0.73 μg/10^6 cells for unmodified DNA.
- Internalized DNA fragments underwent partial 3' end hydrolysis (30-60 nucleotides) and circularization.
Conclusions:
- Murine Krebs-2 cells exhibit native internalization of extracellular DNA, affecting DNA repair processes.
- Modified DNA, particularly with bulky adducts, can inhibit nucleotide excision repair in these cells.
- Characterization of internalization parameters provides insights into DNA-drug interactions and potential therapeutic strategies.
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