Volume 272, Number 39,
Issue of September 26, 1997
pp. 24159-24164
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Ubiquitin-dependent Destruction of Topoisomerase I
Is Stimulated by the Antitumor Drug Camptothecin
(Received for publication, June 9, 1997)
Shyamal D.
Desai
,
Leroy F.
Liu
§
,
Dolores
Vazquez-Abad
¶
and
Peter
D'Arpa
§
From the 
Department of Biochemistry and Molecular Biology,
Uniformed Services University of the Health Sciences, Bethesda,
Maryland 20814-4799, the 
Department of Pharmacology,
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson
Medical School, Piscataway, New Jersey 08854-5635, the § The
Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, and the ¶ Division of Rheumatic Diseases, Department of Medicine,
University of Connecticut School of Medicine,
Farmington, Connecticut 06030
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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