We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.

The major types of DNA damage induced by sunlight in the scope. This latter method allows us to reconstruct three-diskin are DNA photoproducts, such as cyclobutane pyrimidine mensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and hisdimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isotology in multilayered epidermis. Using those techniques, one mers of 6-4PPs. A sensitive method for quantitating and can determine the induction and repair of these three distinct visualizing each type of DNA photoproduct induced by biologtypes of DNA photoproducts in cultured cells and in the skin ically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological efexposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techfects. We have established monoclonal antibodies specific for niques and antibodies, we describe the DNA repair kinetics CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodpigmented cells and in human epidermis. ies with in situ laser cytometry to visualize and measure DNA

We have recently developed a micropore ultraviolet irradiation technique. An isopore membrane filter with 3 microm diameter pores shields ultraviolet C radiation from cultured human fibroblasts, leading to partial irradiation within the cells with an average of about three exposed areas per nucleus. This study addressed the question of whether the spatial distribution of DNA damage within a cell nucleus is important in triggering ultraviolet-induced cytotoxicity. We have examined whether there are differences in cytotoxicity between partially ultraviolet-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 an enzyme-linked immunosorbent assay. We found that 5 J per m2 ultraviolet irradiation produced an equivalent amount of cyclobutane pyrimidine dimers and (6-4) photoproducts per cell as 100 J per m2 with the membrane filter shield. At those doses, we found that both types of ultraviolet irradiation induced similar levels of cytotoxicity as assessed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Both types of ultraviolet-irradiated cells also had similar cell-cycle distribution and apoptosis as measured by flow cytometry. Moreover, no significant differences in repair kinetics for either type of photolesion were observed between the two different ultraviolet 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 ultraviolet-induced cytotoxicity.

A photosensitive form of trichothiodystrophy (TTD) results from mutations in the same XPD gene as the DNA-repair-deficient genetic disorder xeroderma pigmentosum group D (XP-D). Nevertheless, unlike XP, no increase in skin cancers appears in patients with TTD. Although the ability to repair ultraviolet (UV)-induced DNA damage has been examined to explain their cancer-free phenotype, the information accumulated to date is contradictory. In this study, we determined the repair kinetics of cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts (6-4PP) in three TTD cell strains using an enzyme-linked immunosorbent assay. We found that all three TTD cell strains are deficient in the repair of CPD and of 6-4PP. UV sensitivity correlated well with the severity of repair defects. Moreover, accumulation of repair proteins (XPB and proliferating cell nuclear antigen) at localized DNA damage sites, detected using micropore UV irradiation combined with fluorescent antibody labeling, reflected their DNA repair activity. Importantly, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA damage sites and the TFIIH expression. Our results suggest that there is no major difference in the repair defect between TTD and XP-D and that the cancer-free phenotype in TTD is unrelated to a DNA repair defect.

We describe a patient with systemic lupus erythematosus (SLE) associated with topoisomerase I (topo I, Scl-70) antibody, a specific marker for systemic sclerosis (SSc). SSc patients who produce this antibody have severe cutaneous and visceral involvement, and eventually have a poor prognosis. It is rare to find this antibody in patients with other collagen diseases. Only four SLE patients have been reported in the English language literature who were topo I antibody-positive but had no clinical evidence of scleroderma. The serum of our patient with SLE had precipitating topo I antibody from the onset of his disease. Twelve years after the onset of SLE, he developed proximal scleroderma and pulmonary fibrosis. This case reconfirms the prognostic significance of topo I antibody as a predictive marker, and indicates that SLE patients with topo I antibody require careful follow-up for future development of scleroderma.