title: Evidence for different mechanisms of 'unhooking' for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples.
abstract: BACKGROUND
  DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the 'unhooking' step is common to all ICLs.
  
  
  METHODS
  Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment.
  
  
  RESULTS
  Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses.
  
  
  CONCLUSIONS
  These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are 'unhooked' in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.
authors.affiliation: CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Spanswick
authors.forename: Helen L
authors.initials: HL
authors.lastname: Lowe
authors.forename: Claire
authors.initials: C
authors.lastname: Newton
authors.forename: John P
authors.initials: JP
authors.lastname: Bingham
authors.forename: Alessia
authors.initials: A
authors.lastname: Bagnobianchi
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Kiakos
authors.forename: Charles
authors.initials: C
authors.lastname: Craddock
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Ledermann
authors.forename: Daniel
authors.initials: D
authors.lastname: Hochhauser
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005260:Female; D006657:Histones / Q000235:genetics; D059765:Homologous Recombination / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008558:Melphalan / Q000494:pharmacology*; D009101:Multiple Myeloma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics; D015398:Signal Transduction; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; C468783:H2AX protein, human; D006657:Histones; D003841:Deoxycytidine; D004247:DNA; D051135:Rad51 Recombinase; D002945:Cisplatin; D008558:Melphalan; D000093542:Gemcitabine