title: Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide.
abstract: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole.
  
  
  
  DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®.
  
  
  
  Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage.
  
  
  
  Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents.
  
  
  
  Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
authors.forename: Luke
authors.initials: L
authors.lastname: Pett
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Kiakos
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Vijay
authors.initials: V
authors.lastname: Satam
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Pravin
authors.initials: P
authors.lastname: Patil
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Sarah
authors.initials: S
authors.lastname: Laughlin-Toth
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Gregory
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Bowerman
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Olson
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Mia
authors.initials: M
authors.lastname: Savagian
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States.
authors.forename: Megan
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Hope College, Holland, MI 49423, United States; Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Moses
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: W David
authors.initials: WD
authors.lastname: Wilson
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hochhauser
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK. Electronic address: john.hartley@ucl.ac.uk.
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005047:Etoposide / Q000009:adverse effects / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D009757:Nylons / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic
chemical_list: D000951:Antigens, Neoplasm; D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D009757:Nylons; D005047:Etoposide; D004250:DNA Topoisomerases, Type II
keywords: Chemosensitisation; DNA-binding polyamides; Gene modulation; NF-Y; Sequence selectivity; Topoisomerase IIα (topo IIα); Transcription factor-DNA interactions