title: New Topoisomerase I mutations are associated with resistance to camptothecin.
abstract: BACKGROUND
  Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.
  
  
  METHODS
  We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.
  
  
  RESULTS
  In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.
  
  
  CONCLUSIONS
  These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.
authors: Institut de Recherche en Cancérologie de Montpellier, INSERM, Université Montpellier1, CRLC Val d'Aurelle Paul Lamarque, Montpellier, France.
authors: Céline
authors: C
authors: Gongora
authors: Nadia
authors: N
authors: Vezzio-Vie
authors: Sandie
authors: S
authors: Tuduri
authors: Vincent
authors: V
authors: Denis
authors: Annick
authors: A
authors: Causse
authors: Céline
authors: C
authors: Auzanneau
authors: Gwenaëlle
authors: G
authors: Collod-Beroud
authors: Arnaud
authors: A
authors: Coquelle
authors: Philippe
authors: P
authors: Pasero
authors: Philippe
authors: P
authors: Pourquier
authors: Pierre
authors: P
authors: Martineau
authors: Maguy
authors: M
authors: Del Rio
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