Transcriptional regulation of topoisomerase II alpha at confluence and pharmacological modulation of expression by bis-benzimidazole drugs.
Topoisomerase II alpha is a critical gene involved in DNA replication and maintenance of genomic stability. Several chemotherapeutic agents target topoisomerase II and levels of expression are an important factor in chemosensitivity. Transcriptional regulation has been demonstrated to regulate topoisomerase II alpha levels under several circumstances, including cellular confluence, heat shock, and expression of oncogenes including ras and myb. Expression of topoisomerase II alpha is regulated by cellular proliferation; transcriptional down-regulation in confluent cells is modulated through sequences within the promoter. In this study, we examined DNA-protein interactions within the topoisomerase II alpha promoter in exponential and confluent phase NIH3T3 cells. Using electrophoretic mobility shift assay and in vitro DNase I footprint experiments, the involvement of NF-Y in transcriptional regulation was established. Incubation of the DNA minor groove-binding agents Hoechst 33342 and Hoechst 33258 with nuclear extracts revealed drug binding to regions surrounding the inverted CCAAT boxes within the topoisomerase II alpha promoter and displacement of proteins binding to these elements. Addition of both Hoechst 33342 and Hoechst 33258 to NIH3T3 cells at confluence resulted in increased expression of topoisomerase II alpha. In addition, MTT cytotoxicity assays in confluent cells showed an additive effect of incubation with Hoechst 33342 and the topoisomerase II alpha poison etoposide. Therefore, DNA binding drugs which block transcription factor activation of the promoter may deregulate topoisomerase II alpha and this strategy may be of value in modifying gene expression and modulating chemosensitivity.
Department of Oncology, Royal Free and University College Medical School, University College London, Gower Street Campus, London, United Kingdom.
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Tolner
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Hartley
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Hochhauser
D016475:3T3 Cells; D000818:Animals; D000951:Antigens, Neoplasm; D001562:Benzimidazoles / Q000494:pharmacology*; D006690:Bisbenzimidazole / Q000494:pharmacology; D002460:Cell Line; D018983:DNA Footprinting; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004214:Distamycins / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics
C121196:AR-1-144; D000951:Antigens, Neoplasm; D001562:Benzimidazoles; D004268:DNA-Binding Proteins; D004214:Distamycins; D007527:Isoenzymes; D019384:Nucleic Acid Synthesis Inhibitors; D005047:Etoposide; C030000:stallimycin; D004250:DNA Topoisomerases, Type II; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride