Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Camptothecin (CPT) has been shown to induce protein-linked DNA breaks (PLDB), which are stabilized intermediates of topoisomerase I (TOP1) activity. Due to the reversible nature of PLDB and the need for replication fork movement for CPT toxicity, both the time of CPT exposure and TOP1 levels are determinants of CPT toxicity. Therefore, the effects of CPT exposure on TOP1 over time were examined in an established human cell line, KB. Using an in vivo KCl-SDS co-precipitation assay, it was determined that 1 hr of CPT exposure resulted in a concentration-dependent increase in PLDB that reached a maximum at 5 microM CPT. However, prolonged incubations with CPT revealed a concentration- and time-dependent decrease in CPT-induced PLDB formation. The most rapid loss of PLDB was within 6 hr. Neither aphidicolin nor cycloheximide cotreatments altered the PLDB decrease induced by CPT. Immunoblot analysis revealed a reduction in TOP1 protein upon CPT exposure, whereas RNA analysis revealed no changes, which suggested a post-transciptional mechanism of TOP1 down-regulation. The CPT-induced reduction was specific for TOP1, because actin and tubulin levels were unaltered by CPT exposure. Finally, clonogenic assays revealed a small but statistically significant decrease in CPT toxicity throughout the CPT exposure period. Because PLDB formation based on TOP1 levels is an important step in the toxicity of CPT, the CPT-induced TOP1 reduction could be a transient mechanism of resistance for cells to avoid toxic levels of PLDB.
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PMID:Camptothecin induction of a time- and concentration-dependent decrease of topoisomerase I and its implication in camptothecin activity. 753 95

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10-16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction.
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PMID:Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin. 930 65