Identification of an intracellular precursor to DNA excreted by human lymphocytes

Abstract

Phytohemagglutinin-stimulated human peripheral blood lymphocytes in vitro synthesize DNA that is excreted into the culture medium. When such cells are pulse-labeled with [3H]thymidine during the peak of DNA synthesis on day 3 of culture, then cultured for 3 more days in the absence of isotope, labeled DNA moves slowly into the Hirt supernatant cell fraction from the pellet fraction containing chromosomal DNA,and then into the culture medium. The number of copies of excreted DNA sequences in the Hirt pellet fraction was determined for lymphocytes harvested on days 3,4, and 6 after stimulation and compared to the number found in resting lymphocyte DNA and in placenta DNA. While resting lymphocyte and placenta DNAs contain one to two copies of sequences similar to excreted DNA per haploid genome, stimulated lymphocytes on days 3 and 4 of culture contain 3- to 4-fold more copies; by day 6 of culture, stimulated lymphocytes contain only 1- to 2-fold more copies than resting lymphocytes. Thus, phytohemagglutinin induces lymphocytes to selectively replicate several copies of a limited portion of their genome, copies which are then excreted into the culture medium. As determined by reassociation kinetics analysis, a high-molecular-weight DNA fraction from the Hirt supernatant contains sequences found in excreted DNA. This DNA may represent an intermediate formed prior to release of excreted sequences from the cells.