ISSN:
1432-1335
Keywords:
Key words Immunohistochemistry
;
DNA repair
;
Radiation-inducible response
;
Apoptosis
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bax protein in normal and excision-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiation of cells with γ rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bax were monitored using antisera raised against the human proteins. In addition, topoisomerases IIα and IIβ were made visible with specific antibodies. In untreated cells, DNA topoisomerase I was found to occur in the cytoplasm and in nucleoli. Irradiation with γ rays (2–12 Gy) or UV light (0.3–1.2 mW/cm2) changed the staining pattern in nuclei such that a multitude of small topoisomerase-I-rich centers occurred, which were evenly distributed over the karyoplasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6–100 μM concentrations) resulted in similar alterations although the changes were much more pronounced. Combinations of topotecan and γ irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence intensity of the latter may reflect three biological processes: (i) enhanced transcriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separate assays using normal and XP cells, a dose-dependent increase in protein reacting with Bax antibody was observed in nuclei, following treatment with γ rays or topotecan. In addition, topotecan induced a netlike arrangement of this Bax protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4′,6-diamidino-2-phenylindole dihydrochloride revealed that the vesicle-type structures contained DNA. Upon further incubation with topotecan, cells showing the netlike Bax arrangement eventually died. We conclude that topotecan-induced changes made visible by nuclear Bax protein are associated with apoptosis. XP cells, when treated with topotecan, responded more readily than normal cells with both an increase in nuclear Bax protein and rearrangement of Bax, indicating that UV repair functions may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases IIα and IIβ in γ-irradiated cells with antibodies revealed a dramatic increase in the IIα form and a redistribution of the IIβ form representing fragmentation of nucleoli.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s004320050263
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