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Checkpoint Kinase 1 Down-Regulation by an Inducible Small Interfering RNA Expression System Sensitized In vivo Tumors to Treatment with 5-Fluorouracil

Authors' Affiliations: ¹ Laboratory of Molecular Pharmacology, Department of Oncology, and ² Laboratory of Antitumoral Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy

Requests for reprints: Giovanna Damia, Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," via La Masa 19, 20156 Milan, Italy. Phone: 39-2-39014473; Fax: 39-2-3546277; E-mail: damia{at}marionegri.it.

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 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.