Formation of DNA strand breaks in UV-irradiated human fibroblast preparations

Abstract

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m²; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m². Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblasts strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m² indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m². Moreover, after 25 or 50 J/m², strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scrapping. Permeabilization in situ using saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m², UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.