Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIα expression☆

Abstract

1 DNA 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 IIα and topo IIβ. In MCF-7 cells, the half-life (mean ± SEM) values of topo IIα and topo IIβ in situ were 6.6 ± 0.3 and 17.6 ± 2.3 hr, respectively, as determined by [ 35S]methionine/cysteine pulse-chase analysis. Degradation of topo IIα 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 ≅ lactacystin. ATP-dependent degradation of topo IIα, but not topo IIβ, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIα 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 IIα, but not topo IIβ, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the α isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIα appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated–topo IIα 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 IIα expression.