Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase II alpha expression

DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topolo gy and also serves as a target to mediate the cytotoxicity of several antin eoplastic agents. Several reports have demonstrated that a reduction of top o II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo II alpha and topo II beta. In MCF -7 cells, the half-life (mean +/- SEM) values of topo II alpha and topo II beta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determi ned by [S-35]methionine/cysteine pulse-chase analysis. Degradation of topo II alpha in situ was abrogated by the presence of proteasome inhibitors, an d the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent to lactacy stin. ATP-dependent degradation of topo II alpha, but not topo II beta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furt hermore, degradation of topo II alpha was abrogated by the proteasome inhib itors MG132 and MG115, but not by lactacystin, in extracts of asynchronousl y dividing MCF-7 cells. Finally, degradation of topo II alpha, but not topo II beta, was observed to occur in a cell cycle-dependent fashion, in extra cts of synchronized HeLa cells, with maximal loss of the cu isoform occurri ng 2 hr after release from mitotic arrest. This degradation of topo II alph a appeared to be facilitated by an ATP-dependent activity. Furthermore, hig h molecular weight bands (>200 kDa), which may represent polyubiquitinated- topo II alpha conjugates, were also detected in extracts of synchronized He La cells. This study provides evidence for a role of the ubiquitin-proteaso me pathway in the cell cycle-dependent regulation of topo II alpha expressi on. (C) 2001 Elsevier Science Inc. All rights reserved.