Double-Stranded DNA Fragments Bearing Unrepairable Lesions and Their Internalization into Mouse Krebs-2 Carcinoma Cells

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.