Abstract

DNA topoisomerase (topo) IIα gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better understand the mechanisms by which topoIIα expression levels are modulated, we examined topoIIα transcriptional regulation in ICRF-187-sensitive and ICRF-187-resistant human leukemic cell lines that express an increased amount of topoIIα protein and mRNA. Transient transfections of luciferase reporter plasmids containing either the full-length human topoIIα promoter or fragments of it revealed that topoIIα transcriptional activity was significantly increased in the drug-resistant CEM/ICRF-8 cells, compared with CEM cells. Specifically, the transcriptional activity of the full-length topoIIα promoter (nucleotides −557 to +90) was doubled in CEM/ICRF-8 compared with CEM cells. Serial deletion of the topoIIα promoter permitted localization of the region responsible for its up-regulation in the drug-resistant cells between nucleotides −557 and −162, which includes the last three inverted CCAAT elements (ICE) 3 to 5. Note that construction of a point mutation in ICE3 resulted in a significant increase in transcriptional activity of the topoIIα promoter in the drug-sensitive CEM cells. In addition, by electrophoretic mobility shift assay, ICE3 was recognized by a protein complex containing NF-YB that was present at reduced levels in the topoIIα-overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoIIα gene expression. This is the first study to show that topoIIα transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoIIα promoter.