Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response*
- From the ‡Laboratory of Molecular Pharmacology and
- the §Gene Silencing Section, Genetics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
- 1To whom correspondence should be addressed: Bldg. 37, Rm. 5068, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-5944; Fax: 301-480-4988; E-mail: pommier{at}nih.gov.
Abstract
To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by histone γ-H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels and induces the nuclear translocation of RRM2. The checkpoint kinase 1 (Chk1) was up-regulated and activated in response to CPT, and CHEK1 down-regulation by siRNA and small molecule inhibitors of Chk1 blocked RRM2 induction by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs and DNA repair. Specifically, we propose that upon DNA damage, Chk1 activation, mediated by ATM and ATR, up-regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 expression levels coupled with its nuclear recruitment suggests an active role for ribonucleotide reductase in the cellular response to CPT-mediated DNA damage that could potentially be exploited as a strategy for enhancing the efficacy of topoisomerase I inhibitors.
Footnotes
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↵* This research was supported, in whole or in part, by the National Institutes of Health Intramural Research Program, Center for Cancer Research, NCI.
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↵
The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.
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↵2 The abbreviations used are:
- Top1
-
topoisomerase I
- ATM
-
ataxia-telangiectasia (AT) mutated
- ATR
-
ataxia-telangiectasia and Rad3-related
- CPT
-
camptothecin
- Chk1
-
checkpoint kinase 1
- E2F1
-
E2F transcription factor 1
- RNR
-
ribonucleotide reductase
- RRM2
-
ribonucleotide reductase R2
- siRNA
-
small interfering RNA
- BrdUrd
-
bromodeoxyuridine
- PBS
-
phosphate-buffered saline
- PI
-
propidium iodide
- HU
-
hydroxyurea.
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- Received April 2, 2009.
- Revision received May 4, 2009.
