The Total Amount of DNA Damage Determines Ultraviolet-Radiation-Induced Cytotoxicity after Uniform- or Localized Irradiation of Human Cells

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We have recently developed a micropore ultraviolet (UV) irradiation technique. An isopore membrane filter with 3μm diameter pores shields UVC radiation from cultured human fibroblasts, leading to partial irradiation within the cells with an average of about 3 exposed areas per nucleus. The present study addressed the question of whether the spatial distribution of DNA damage within a cell nucleus is important in triggering UV-induce cytotoxicity. We have examined whether there are differences incytotoxicity between partially UV-irradiated cells and uniformly irradiated cells after equal amounts of DNA damage were induced in the cell nuclei. We first determined DNA damage formation in normal human fibroblasts using anenzyme-linked immunosorbent assay. We found that 5 J per m^2 UV irradiation produced an equivalent amount of cyclobutane pyrimidine dimers and (6-4) photoproducts per cell as 100 J per m^2 with the membrane filter shield. At those doses, we found that both types of UV irradiation induced similar levels of cytotoxicity as assessed by an MTS assay. Both types of UV irradiated cells also had similar cell-cycle distribution and apoptosis as measured by flowcytometry. Moreover, no significant differences in repair kinetics for either type of photolesion were observed between the two different UV treatments. Similar results were obtained in Cockayne syndrome cells that are defective in transcription-coupled nucleotide excision repair Present results indicate that in the range of photoproducts studied, the spatial distribution of DNA damage within a cell is less important than the amount of damage in triggering UV-induced cytotoxicity.