title: Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate.
abstract: BACKGROUND
  Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29.
  
  
  METHODS
  Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry.
  
  
  RESULTS
  DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV.
  
  
  CONCLUSIONS
  Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.
authors.affiliation: Research center, Division of Digestive and Oncologic Surgery, Ambroise Pare Hospital and University of Versailles- Saint-Quentin, Boulogne, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Finzi
authors.forename: Aurore
authors.initials: A
authors.lastname: Kraemer
authors.forename: Claude
authors.initials: C
authors.lastname: Capron
authors.forename: Severine
authors.initials: S
authors.lastname: Noullet
authors.forename: Diane
authors.initials: D
authors.lastname: Goere
authors.forename: Christophe
authors.initials: C
authors.lastname: Penna
authors.forename: Bernard
authors.initials: B
authors.lastname: Nordlinger
authors.forename: Josette
authors.initials: J
authors.lastname: Legagneux
authors.forename: Jean-Fançois
authors.initials: JF
authors.lastname: Emile
authors.forename: Robert
authors.initials: R
authors.lastname: Malafosse
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000628:therapy; D003561:Cytarabine / Q000494:pharmacology; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D000998:Antiviral Agents; D003561:Cytarabine; D016590:Aphidicolin; D013937:Thymidine Kinase; D015774:Ganciclovir; D008727:Methotrexate