Spontaneous extracellular synthesis of DNA released by human blood lymphocytes

Abstract

Human lymphocytes were shown to release, in vitro and in the absence of any stimulation, a complex containing DNA. It has also been reported that the release process is unrelated to cell death and is regulated by a homeostatic mechanism. Some properties of the extracellular DNA were investigated. When a phosphorylated precursor was added to the cell-free supernatant, the DNA recovered from the medium was labeled. Evidence that DNA lebeling represented true precursor incorporation and not simple attachment was obtained from nearest neighbor analysis data. When [alpha-32P]thymidine triphosphate was added to the supernatant and the labeled DNA was completely hydrolyzed to 3'-deoxyribonucleotides, radioactivity was found in all four nucleotides. Although the exact kind of synthesis cannot be determined at this stage, the possibility of a terminal transferase system in which the enzyme would merely add a nucleotide at the end of the chain was eliminated since comparative digestion with DNase and venom phosphodiesterase showed that labeling was located along the whole length of the chain. Precursor incorporation into the DNA was inhibited by DNase, RNase, Pronase, and actinomycin D. This extracellular synthesis was not affected by cell death rate. The renaturation curve of the extracellular [3H]DNA synthesized in the cell-free medium showed a lack of gene reiteration suggesting a preferential synthesis of unique sequences.