Studies on DNA Release by Cultured Rat Lymphoblasts

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Abstract
Rat lymphoblasts induced by concanavalin A (Con A) and cultured in the absence of lectin under serum-free conditions for 3 days showed a 2- to 3-fold increase in cell number and total amount of DNA per culture. However, at the end of the culture, 20–30% of the DNA was present in the medium. 3H-thymidine or 3H-bromodeoxyuridine label incorporated into DNA during a pulse at the end of the Con A stimulation period (60–65 h) was also released by the blast cells and represented, after 3 days of further culture, 40–55% of the intracellular counts measured at the end of the pulse. Blast cells cultured in the continued presence of Con A showed similar patterns of proliferation and total DNA increase, but only 18% of the labelled thymidine was released during the 3 days. By neutral sucrose gradient analysis, material released in absence of mitogen could be shown to consist of a DNA-protein complex. The DNA was characterized as a double-stranded molecule with an average sedimentation value of 9–10 S. The DNA released from cells pulsed with labelled thymidine or bromodeoxyuridine increased in specific activity, whereas the specific activity of cellular DNA decreased with time. CsC1 gradient analysis showed that the released DNA maintains the same density throughout the duration of the culture, whereas cellular DNA exhibits a shift towards a lighter density. It is suggested that a significant part of the DNA synthesized during blast transformation subserves immunologic functions of the blast rather than cell replication and is discarded as the blasts return to a small cell configuration.