Role of an inverted CCAAT element in human topoisomerase II alpha gene expression in ICRF-187-sensitive and -resistant CEM leukemic cells

DNA topoisomerase (topo) II alpha gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better und erstand the mechanisms by which topoII a expression levels are modulated, w e examined topoII a transcriptional regulation in ICRF-187-sensitive and IC RF-187-resistant human leukemic cell lines that express an increased amount of topoII alpha protein and mRNA. Transient transfections of luciferase re porter plasmids containing either the full-length human topoII alpha promot er or fragments of it revealed that topoII alpha transcriptional activity w as significantly increased in the drug-resistant CEM/ICRF-8 cells, compared with CEM cells. Specifically, the transcriptional activity of the full-len gth topoII alpha promoter (nucleotides -557 to +90) was doubled in CEM/ICRF -8 compared with CEM cells. Serial deletion of the topoII alpha promoter pe rmitted 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 transcripti onal activity of the topoII alpha 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 alpha -overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoII alpha gene expression. Th is is the first study to show that topoII alpha transcriptional up-regulati on in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoII alpha promoter.