title: Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil.
abstract: OBJECTIVE
  After DNA damage, checkpoints pathways are activated in the cells to halt the cell cycle, thus ensuring repair or inducing cell death. To better investigate the role of checkpoint kinase 1 (Chk1) in cellular response to different anticancer agents, Chk1 was knocked down in HCT-116 cell line and in its p53-deficient subline by using small interfering RNAs (siRNA).
  
  
  METHODS
  Chk1 was abrogated by transient transfection of specific siRNA against it, and stable tetracycline-inducible Chk1 siRNA clones were obtained transfecting cells with a plasmid expressing two siRNA against Chk1. The validated inducible system was then translated in an in vivo setting by transplanting the inducible clones in nude mice.
  
  
  RESULTS
  Transient Chk1 down-regulation sensitized HCT-116 cells, p53-/- more than the p53 wild-type counterpart, to DNA-damaging agents 5-fluorouracil (5-FU), doxorubicin, and etoposide treatments, with no modification of Taxol and PS341 cytotoxic activities. Inhibition of Chk1 protein levels in inducible clones on induction with doxycycline correlated with an increased cisplatin and 5-FU activity. Such effect was more evident in a p53-deficient background. These clones were transplanted in nude mice and a clear Chk1 down-regulation was shown in tumor samples of mice given tetracycline in the drinking water by immunohistochemical detection of Chk1 protein. More importantly, an increased 5-FU antitumor activity was found in tumors with the double Chk1 and p53 silencing.
  
  
  CONCLUSIONS
  These findings corroborate the fact that Chk1 protein is a molecular target to be inhibited in tumors with a defective G1 checkpoint to increase the selectivity of anticancer treatments.
authors.affiliation: Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Ganzinelli
authors.forename: Laura
authors.initials: L
authors.lastname: Carrassa
authors.forename: Francesca
authors.initials: F
authors.lastname: Crippa
authors.forename: Michele
authors.initials: M
authors.lastname: Tavecchio
authors.forename: Massimo
authors.initials: M
authors.lastname: Broggini
authors.forename: Giovanna
authors.initials: G
authors.lastname: Damia
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015153:Blotting, Western; D001897:Boronic Acids; D000069286:Bortezomib; D002453:Cell Cycle / Q000502:physiology*; D045744:Cell Line, Tumor / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D005472:Fluorouracil / Q000627:therapeutic use*; D016158:Genes, p53; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011719:Pyrazines; D034741:RNA, Small Interfering; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D011719:Pyrazines; D034741:RNA, Small Interfering; D000069286:Bortezomib; D005047:Etoposide; D004317:Doxorubicin; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D017239:Paclitaxel; D005472:Fluorouracil