Detection of laser-UV microirradiation-induced DNA photolesions by immunofluorescent staining

A low-power laser-UV microbeam of wave-length 257 nm was used for microirradiation of a small part of the nucleus of Chinese hamster cells. Following fixation in interphase or in the subsequent metaphase indirect immunofluorescent staining was performed with antiserum to photoproducts of DNA treated with far UV light. The results show that antibodies specific for UV-irradiated DNA can be used for a direct detection of laser-UV microirradiation-induced DNA photolesions. The potential usefulness of this method for investigation of the spatial arrangement of chromosomes in the interphase nucleus is discussed

Effects of Laser UV-Microirradiation (λ = 2573 A) on Proliferation of Chinese Hamster Cells

A laser uv-microbeam with a wavelength of 2573 Å having a minimum spot diameter of approx 0.5 μm was used to microirradiate interphase cells of a V-79 subline of Chinese hamster cells. The incident energy necessary to induce a significant decrease of proliferation was 30 to 60 times larger after microirradiation of cytoplasm as compared with microirradiation of nucleoplasm. The mean value of relative cell numbers 40 hr after irradiation as a function of incident energy did not differ whether the cells were microirradiated lying singly or together in small groups. Analysis of individual growth curves of singly lying cells microirradiated in the nucleoplasm with the same energy showed heterogeneous reactions. The incident energy per cell compatible with proliferation of about 50% of the cells after microirradiation of nucleoplasm was approx. 2× 10sup-3/sup ergs. From this value it is suggested that the energy density within the focus was in the region of several thousand ergs per square millimeter. Photochemical effects are thought to be the cause of growth disturbance, while thermal effects are excluded

Laser Microirradiation of Chinese Hamster Cells at Wavelength 365 nm

Cells of a V79 subline of the Chinese hamster were microirradiated at wavelength 365 nm in the presence of the psoralen derivative, trioxsalen. Microirradiation was accomplished by a pulsed argon laser microbeam either in anaphase or in interphase 3 hr after mitosis. Inhibition of clonal growth and formation of micronuclei at the first postirradiation mitosis were observed after microirradiation of anaphase chromosomes and of small parts of the interphase nucleus. Microirradiation of the cytoplasm beside the interphase nucleus or between the sets of chromosomes moving apart from each other in anaphase did not produce these effects. Anaphase experiments showed that only the daughter cell which received microirradiated chromatin exhibited an abnormal growth pattern. Most interestingly, shattering of the whole chromosome complement could be induced by microirradiation of small parts of the interphase nucleus and post-treatment with caffeine. Since microirradiation of chromatin in the absence of psoralen was not effective, we consider formation of psoralen photoadducts to nucleic acids in microirradiated chromatin to be the specific cause of the effects. We suggest that DNA photolesions in chromosome segments present in the microirradiated part of the nucleus can induce shattering of all the chromosomes in the microirradiated nucleus. Several possibilities are discussed to explain this unexpected finding