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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

Ganzinelli, Monica; Carrassa, Laura; Crippa, Francesca; Tavecchio, Michele LU ; Broggini, Massimo and Damia, Giovanna (2008) In Clinical Cancer Research 14(16). p.41-5131
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... (More)

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.

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published
keywords
Animals, Antineoplastic Agents, Blotting, Western, Boronic Acids, Bortezomib, Cell Cycle, Cell Line, Tumor, Checkpoint Kinase 1, Down-Regulation, Doxorubicin, Etoposide, Female, Fluorescent Antibody Technique, Fluorouracil, Genes, p53, Humans, Immunohistochemistry, Mice, Mice, Nude, Neoplasms, Experimental, Paclitaxel, Protein Kinases, Pyrazines, RNA, Small Interfering, Transfection, Xenograft Model Antitumor Assays, Journal Article, Research Support, Non-U.S. Gov't
in
Clinical Cancer Research
volume
14
issue
16
pages
41 - 5131
publisher
American Association for Cancer Research
external identifiers
  • scopus:52649127811
ISSN
1078-0432
DOI
10.1158/1078-0432.CCR-08-0304
language
English
LU publication?
no
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b566bfac-769f-4a94-a0ba-8c3543624357
date added to LUP
2017-03-07 09:13:35
date last changed
2017-10-08 04:59:43
@article{b566bfac-769f-4a94-a0ba-8c3543624357,
  abstract     = {<p>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).</p><p>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.</p><p>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.</p><p>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.</p>},
  author       = {Ganzinelli, Monica and Carrassa, Laura and Crippa, Francesca and Tavecchio, Michele and Broggini, Massimo and Damia, Giovanna},
  issn         = {1078-0432},
  keyword      = {Animals,Antineoplastic Agents,Blotting, Western,Boronic Acids,Bortezomib,Cell Cycle,Cell Line, Tumor,Checkpoint Kinase 1,Down-Regulation,Doxorubicin,Etoposide,Female,Fluorescent Antibody Technique,Fluorouracil,Genes, p53,Humans,Immunohistochemistry,Mice,Mice, Nude,Neoplasms, Experimental,Paclitaxel,Protein Kinases,Pyrazines,RNA, Small Interfering,Transfection,Xenograft Model Antitumor Assays,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {08},
  number       = {16},
  pages        = {41--5131},
  publisher    = {American Association for Cancer Research},
  series       = {Clinical Cancer Research},
  title        = {Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil},
  url          = {http://dx.doi.org/10.1158/1078-0432.CCR-08-0304},
  volume       = {14},
  year         = {2008},
}