Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks.
Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.
Boerrigter
M E
ME
TNO Institute for Experimental Gerontology, Rijswijk, The Netherlands.
Mullaart
E
E
van der Schans
G P
GP
Vijg
J
J
D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000032:analysis / Q000528:radiation effects*; D005720:Gamma Rays; D006801:Humans; D007399:Interphase / Q000528:radiation effects*; D008214:Lymphocytes / Q000378:metabolism / Q000502:physiology / Q000528:radiation effects*
D004277:DNA, Single-Stranded