Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil

Monica Ganzinelli, Laura Carrassa, Francesca Crippa, Michele Tavecchio, Massimo Broggini, Giovanna Damia

Research output: Contribution to journalArticle

Abstract

Purpose: 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). Experimental Design: 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 etopo-side 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 cis-platin 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 G 1checkpoint to increase the selectivity of anticancer treatments.

Original languageEnglish
Pages (from-to)5131-5141
Number of pages11
JournalClinical Cancer Research
Volume14
Issue number16
DOIs
Publication statusPublished - Aug 15 2008

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ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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