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Role of an inverted CCAAT element in human topoisomerase IIalpha gene expression in ICRF-187-sensitive and -resistant

S E Morgan1, W T Beck

  • 1Division of Molecular Pharmacology, Departments of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.

Molecular Pharmacology
|February 13, 2001
PubMed

Insights

Drug resistance in leukemia involves altered DNA topoisomerase IIalpha (topoIIalpha) gene expression. This study reveals that a specific promoter region, ICE3, negatively regulates topoIIalpha, and its reduced function contributes to drug resistance.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Genetics

Background:

  • Tumor cells resistant to topoisomerase II (topoII) inhibitors often exhibit altered topoIIalpha gene expression.
  • Understanding topoIIalpha transcriptional regulation is crucial for deciphering drug resistance mechanisms.

Purpose of the Study:

  • To investigate the transcriptional regulation of the human DNA topoisomerase IIalpha (topoIIalpha) gene in drug-resistant leukemia cells.
  • To identify specific promoter elements responsible for increased topoIIalpha expression in cells resistant to topoII inhibitors.

Main Methods:

  • Transient transfection assays using luciferase reporter plasmids with human topoIIalpha promoter constructs.
  • Analysis of promoter activity in drug-sensitive (CEM) and drug-resistant (CEM/ICRF-8) human leukemic cell lines.
  • Electrophoretic mobility shift assays (EMSA) to study protein-DNA interactions within the topoIIalpha promoter.

Main Results:

  • TopoIIalpha transcriptional activity was significantly increased (doubled) in drug-resistant CEM/ICRF-8 cells compared to sensitive CEM cells.
  • A key regulatory region responsible for this up-regulation was localized between nucleotides -557 and -162 of the topoIIalpha promoter, encompassing inverted CCAAT elements (ICE) 3 to 5.
  • A point mutation in ICE3 significantly increased promoter activity in drug-sensitive cells, and EMSA indicated reduced binding of an NF-YB containing complex to ICE3 in resistant cells, suggesting negative regulation by ICE3.

Conclusions:

  • Transcriptional up-regulation of topoIIalpha in ICRF-187-resistant cells is partly mediated by altered regulation of the third inverted CCAAT box (ICE3) in the topoIIalpha promoter.
  • ICE3 appears to function as a negative regulatory element in human topoIIalpha gene expression, and its diminished regulatory capacity contributes to increased topoIIalpha levels in resistant cells.

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