Vol. 59, Issue 2, 203-211, February 2001

Role of an Inverted CCAAT Element in Human Topoisomerase II Gene Expression in ICRF-187-Sensitive and -Resistant CEM Leukemic Cells

Susan E. Morgan¹ and William T. Beck

Division of Molecular Pharmacology, Departments of Molecular Genetics (S.E.M, W.T.B.) and Pharmaceutics and Pharmacodynamics (W.T.B.), University of Illinois at Chicago, Chicago, Illinois

DNA topoisomerase (topo) II gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better understand the mechanisms by which topoII expression levels are modulated, we examined topoII transcriptional regulation in ICRF-187-sensitive and ICRF-187-resistant human leukemic cell lines that express an increased amount of topoII protein and mRNA. Transient transfections of luciferase reporter plasmids containing either the full-length human topoII promoter or fragments of it revealed that topoII 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 topoII promoter (nucleotides 557 to +90) was doubled in CEM/ICRF-8 compared with CEM cells. Serial deletion of the topoII 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 topoII 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 topoII-overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoII gene expression. This is the first study to show that topoII transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoII promoter.