Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIalpha expression.
1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.
Department of Pharmacology, University of Toronto, M5S 3E2, Toronto, Ontario, Canada.
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Salmena
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Lam
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McPherson
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GJ
Goldenberg
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000951:Antigens, Neoplasm; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002457:Cell Extracts; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D007976:Leupeptins; D009097:Multienzyme Complexes; C089486:carbobenzoxy-leucyl-leucyl-norvalinal; C063261:acetylleucyl-leucyl-norleucinal; C067713:lactacystin; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004250:DNA Topoisomerases, Type II; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine