Subnuclear distribution of DNA topoisomerase I and Bax protein in normal and xeroderma pigmentosum fibroblasts after irradiation with UV light and gamma rays or treatment with topotecan

Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bar protein in normal and exci sion-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiat ion of cells with gamma rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bar were monitored using ant isera raised against the human proteins. In addition, topoisomerases II alp ha. and II beta were made visible with specific antibodies. In untreated ce lls, DNA topoisomerase I was found to occur in the cytoplasm and in nucleol i. Irradiation with gamma rays (2-12 Gy) or UV light (0.3-1.2 mW/cm(2)) cha nged the staining pattern in nuclei such that a multitude of small topoisom erase-I-rich centers occurred! which were evenly distributed over the karyo plasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6-100 mu M concentrations) resulted in similar alterations alth ough the changes were much more pronounced. Combinations of topotecan and g amma irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence inte nsity of the latter may reflect three biological processes: (i) enhanced tr anscriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separat e assays using normal and XP cells, a dose-dependent increase in protein re acting with Bar antibody was observed in nuclei, following treatment with g amma rays or topotecan. In addition, topotecan induced a netlike arrangemen t of this Bar protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4',6-diamidino-2-phenylindole dihydrochloride r evealed that the vesicle-type structures contained DNA. Upon further incuba tion with topotecan, cells showing the netlike Bar arrangement eventually d ied. We conclude that topotecan-induced changes made visible by nuclear Bar protein are associated with apoptosis. XP cells, when treated with topotec an, responded more readily than normal cells with both an increase in nucle ar Bar protein and rearrangement of Bar, indicating that UV repair function s may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases II alpha and II beta in gamma-irradiated cells with a ntibodies revealed a dramatic increase in the II alpha form and a redistrib ution of the II beta form representing fragmentation of nucleoli.