Ubiquitin-dependent Destruction of Topoisomerase I Is Stimulated by the Antitumor Drug Camptothecin

Abstract

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. DNA topoisomerase I (TOP1)1 is a vital DNA metabolic enzyme as well as a molecular target of antitumor drugs. TOP1 relaxes DNA supercoils by cleaving a single strand of duplex DNA and passing the complimentary DNA strand though the cleaved strand before religation (1). In a transient reaction intermediate, termed the cleavable complex, the enzyme links covalently to DNA through tyrosine 723 (human TOP1) leaving a DNA break having a free 5Ј hydroxyl end. Stopping the TOP1 reaction with strong protein denaturants produces a very low yield of TOP1 linked covalently to DNA. The rarity of this product is a reflection of the transient nature of the "cleaved" reaction intermediate. Antitumor drugs that act on TOP1 inhibit the DNA religation step through a reversible mechanism. The inhibition of religation can be observed as an increase in the yield of the enzyme-DNA covalent complex pro...

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…Thus, it has been suggested that the Top1 must undergo proteolysis in order for efficient Tdp1 activity [19,41,45], which is in agreement with studies demonstrating that the effectiveness of Tdp1 processing decreases as the length of the Top1 polypeptide is extended [45]. Accordingly, several studies have shown that Top1 is degraded in some cells lines following CPT treatment, suggesting that Top1 ubiquitination and degradation contribute to CPT resistance [46][47][48][49]. In addition, prevention of Top1 degradation by the proteasome inhibitors, MG132 [47] or PS-341 (clinically used) [50], results in increased sensitivity to CPT.…”

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…Note the nibbled colony morphology of slx8Δ and ubc9-1 single mutants. (B) Extracts from the indicated yeast mutants were prepared by the NaOH method [56] and analyzed for Smt3-protein conjugates by 10% SDS-PAGE and immunoblotting using antibodies against Smt3. Identical samples were run on 17% SDS-PAGE, blotted, and probed for free Smt3 or RPA1 as internal loading controls.…”

Mechanisms of resistance to topoisomerase I-targeting drugs

“…Recently, post-translational modifications of Top1 were reported after CPT treatment of cells and may be involved in resistance. Top1 is ubiquitinated and degraded after cells are treated with CPT, which appears to occur in the context of the ternary complex rather than free Top1, since nuclease treatment of cell lysates is necessary to visualize Top1-ubiquitin conjugates by Western blotting (Desai et al, 1997). Recently, tumor cells deficient in CPT-induced Top1 downregulation were found to be more sensitive to CPT, implicating ubiquitination of Top1 as an important determinant of cellular sensitivity (Desai et al, 2001).…”