Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity.

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice.

BACKGROUND: When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment. METHODS: Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls. RESULTS: An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection. CONCLUSION: Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment.

Natural human gene correction by small extracellular genomic DNA fragments.

Classical gene targeting employs natural homologous recombination for a gene correction using a specially designed and artificially delivered DNA construct but the method is very inefficient. On the other hand, small DNA fragments in the form of tiny chromatin-like particles naturally present in blood plasma can spontaneously penetrate into human cells and cell nuclei. We hypothesized that these natural DNA nanoparticles with recombinagenic free ends might be effective agents for gene replacement therapy. We demonstrate that a mixture of small fragments of total human chromatin from non-mutant cells added to a culture medium without transfection agents efficiently repaired a 47 base pair deletion in the CASP3 gene in 30% of treated human MCF7 breast cancer cells, as shown by restoration of caspase-3 apoptotic function and CASP3 DNA and mRNA structure. Such an innate gene replacement mechanism might function naturally in an organism using its own apoptotic DNA fragments. This mechanism might enable human cancer cell phenotype normalization in the presence of excess normal cells.

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell.

BACKGROUND: The blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell. RESULTS: The existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7) cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction) was assessed. The extracellular DNA delivered to the cell is characterized quantitatively. CONCLUSION: It is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor alpha-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.