Sister chromatid exchange (SCE) induced by laser-UV-microirradiation: correlation between the distribution of photolesions and the distribution of SCEs.

Abstract

Small, medium, and large nuclear areas comprising approximately 5, 30, or 80% of the total area of the interphase nuclei of Chinese hamster cells (M3-1) cultivated in vitro were irradiated with a laser-UV-microbeam of wavelength 257 nm. The DNA of the cells was substituted with 5-bromodeoxyuridine (BrdUrd) for 1 cell cycle in one set of experiments. After microirradiation the cells were grown for a second cycle in medium without BrdUrd (protocol A). In a second set, cells with nonsubstituted DNA were microirradiated and grown for 2 additional cycles, the first in the presence, the second in the absence of BrdUrd (protocol B). In situ chromosome preparation and differential chromatid staining was subsequently performed. The induction of sister chromatid exchanges (SCEs) was found to be dependent on both the ultraviolet (UV) dose and the spatial distribution of the UV energy within the cell nucleus. Following both protocols the average number of chromosomes with SCEs was significantly higher after microirradiation of a large nuclear area as compared to microirradiation of a small nuclear area. In the latter case, multiple SCEs were noted on individual chromosome arms at the first postirradiation mitosis (protocol A). In other cells, especially at higher doses, protocol A resulted in shattering of a few closely neighbored chromosomes which were surrounded by intact ones with normal SCE levels. Microirradiation of medium-sized nuclear areas produced high levels of SCEs over a number of chromosomes which still appeared spatially related in a part of the metaphase spread. Finally, high SCE levels could be observed over most or all chromosomes when a large nuclear area (up to 100%) was exposed to the microbeam. Following protocol B the increase of SCEs was much less pronounced. Microirradiation of a small part of the cytoplasm in addition to the nuclei did not induce SCEs. Our results support the concept (i) that interphase chromosomes occupy distinct nuclear domains and indicate (ii) that the induction of SCEs by UV light is restricted to microirradiated chromatin.