Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.
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 gamma-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.
Laboratory of Molecular Pharmacology, Genetics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yong-Wei
YW
Zhang
Tamara L
TL
Jones
Scott E
SE
Martin
Natasha J
NJ
Caplen
Yves
Y
Pommier
D064007:Ataxia Telangiectasia Mutated Proteins; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002166:Camptothecin / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000235:genetics; D000071877:Checkpoint Kinase 1; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D004249:DNA Damage / Q000235:genetics*; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D050687:E2F1 Transcription Factor / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D034741:RNA, Small Interfering; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D059004:Topoisomerase I Inhibitors; D025521:Tumor Suppressor Proteins; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D002166:Camptothecin