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.