Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil
(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.
(Less)
- author
- Ganzinelli, Monica ; Carrassa, Laura ; Crippa, Francesca ; Tavecchio, Michele LU ; Broggini, Massimo and Damia, Giovanna
- publishing date
- 2008-08-15
- type
- Contribution to journal
- publication status
- 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 Inc.
- external identifiers
-
- scopus:52649127811
- pmid:18698031
- ISSN
- 1078-0432
- DOI
- 10.1158/1078-0432.CCR-08-0304
- language
- English
- LU publication?
- no
- id
- b566bfac-769f-4a94-a0ba-8c3543624357
- date added to LUP
- 2017-03-07 09:13:35
- date last changed
- 2025-04-14 17:22:06
@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}},
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}},
language = {{eng}},
month = {{08}},
number = {{16}},
pages = {{41--5131}},
publisher = {{American Association for Cancer Research Inc.}},
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}},
doi = {{10.1158/1078-0432.CCR-08-0304}},
volume = {{14}},
year = {{2008}},
}