Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response
2009; Elsevier BV; Volume: 284; Issue: 27 Linguagem: Inglês
10.1074/jbc.m109.003020
ISSN1083-351X
AutoresYongwei Zhang, Tamara L. Jones, Scott E. Martin, Natasha J. Caplen, Yves Pommier,
Tópico(s)Acute Myeloid Leukemia Research
ResumoTo 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. 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. Two water-soluble DNA topoisomerase 1 (Top1) 2The abbreviations used are: Top1topoisomerase IATMataxia-telangiectasia (AT) mutatedATRataxia-telangiectasia and Rad3-relatedCPTcamptothecinChk1checkpoint kinase 1E2F1E2F transcription factor 1RNRribonucleotide reductaseRRM2ribonucleotide reductase R2siRNAsmall interfering RNABrdUrdbromodeoxyuridinePBSphosphate-buffered salinePIpropidium iodideHUhydroxyurea. inhibitors, derived from camptothecin (CPT), are in clinical use; topotecan, for the treatment of ovarian and lung cancers, and irinotecan, for colorectal cancers. Further CPT derivatives and non-CPT Top1 inhibitors are in preclinical development as anticancer agents (1.Bailly C. Crit. Rev. Oncol. Hematol. 2003; 45: 91-108Crossref PubMed Scopus (102) Google Scholar, 2.Pommier Y. Nat. Rev. Cancer. 2006; 6: 789-802Crossref PubMed Scopus (1650) Google Scholar, 3.Teicher B.A. Biochem. Pharmacol. 2008; 75: 1262-1271Crossref PubMed Scopus (166) Google Scholar). Despite the fact that camptothecins are highly targeted agents with Top1 as their sole cellular target, the response of cancer cells to the inhibition of Top1 by camptothecins is highly variable and remains for the most part undefined (2.Pommier Y. Nat. Rev. Cancer. 2006; 6: 789-802Crossref PubMed Scopus (1650) Google Scholar, 4.Goldwasser F. Shimizu T. Jackman J. Hoki Y. O'Connor P.M. Kohn K.W. Pommier Y. Cancer Res. 1996; 56: 4430-4437PubMed Google Scholar, 5.Li T.K. Liu L.F. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 53-77Crossref PubMed Scopus (475) Google Scholar). One of the critical mechanism for the antiproliferative activity of camptothecins is the generation of replication-associated DNA double-strand breaks by collisions between replication forks and drug-stabilized Top1 cleavage complexes (6.Hsiang Y.H. Lihou M.G. Liu L.F. Cancer Res. 1989; 49: 5077-5082PubMed Google Scholar, 7.Holm C. Covey J.M. Kerrigan D. Pommier Y. Cancer Res. 1989; 49: 6365-6368PubMed Google Scholar), which results in phosphorylation of histone H2AX (8.Bonner W.M. Redon C.E. Dickey J.S. Nakamura A.J. Sedelnikova O.A. Solier S. Pommier Y. Nat. Rev. Cancer. 2008; 8: 957-967Crossref PubMed Scopus (1273) Google Scholar) that can be detected as histone γH2AX foci (9.Furuta T. Takemura H. Liao Z.Y. Aune G.J. Redon C. Sedelnikova O.A. Pilch D.R. Rogakou E.P. Celeste A. Chen H.T. Nussenzweig A. Aladjem M.I. Bonner W.M. Pommier Y. J. Biol. Chem. 2003; 278: 20303-20312Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 10.Seiler J.A. Conti C. Syed A. Aladjem M.I. Pommier Y. Mol. Cell. Biol. 2007; 27: 5806-5818Crossref PubMed Scopus (187) Google Scholar). topoisomerase I ataxia-telangiectasia (AT) mutated ataxia-telangiectasia and Rad3-related camptothecin checkpoint kinase 1 E2F transcription factor 1 ribonucleotide reductase ribonucleotide reductase R2 small interfering RNA bromodeoxyuridine phosphate-buffered saline propidium iodide hydroxyurea. Synthetic siRNA-based RNAi screening is emerging as a powerful approach to revealing the determinants of cellular responses to drugs. Using a synthetic siRNA-based RNAi screen to identify genes whose silencing affects the activity of CPT, we found that RNAi against RRM1 and RRM2, both ribonucleotide reductase genes, markedly enhanced the cytotoxicity of CPT. The ribonucleotide reductase (RNR) enzyme complex is essential for the de novo synthesis of deoxyribonucleotides (dNTPs) precursors for DNA synthesis. RNR catalyzes the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates and maintains a highly regulated and balanced pool of dNTPs for DNA replication and repair. A failure in the control of dNTP levels leads to cell death or genetic abnormalities (11.Nordlund P. Reichard P. Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (842) Google Scholar, 12.Herrick J. Sclavi B. Mol. Microbiol. 2007; 63: 22-34Crossref PubMed Scopus (110) Google Scholar). In mammals, RNR is an heterodimeric tetramer composed of two identical large subunits RRM1 and two identical small subunit RRM2 (11.Nordlund P. Reichard P. Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (842) Google Scholar). Each RRM1 subunit contains an active site (controlling enzyme activity) and an allosteric site (controlling substrate specificity by binding nucleoside triphosphates). Each RRM2 subunits contains a non-heme (binuclear) iron center and a stable tyrosyl free radical. Both are essential for catalysis (11.Nordlund P. Reichard P. Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (842) Google Scholar). Recently, an additional small subunit has been found, RRM2B (p53R2), which is induced by p53 and can substitute for RRM2 to form a highly active RNR complex involved in DNA repair (for review, see Ref. 11.Nordlund P. Reichard P. Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (842) Google Scholar). RNR activity is closely regulated during the cell cycle, peaking in S-phase. In yeast, expression of the large subunit RNR1 fluctuates more than 10-fold during the cell cycle, whereas the small subunit RNR2 mRNA levels show only a 2-fold change. In mammalian cells, RRM2 protein levels begin to rise in late G1 and reach their highest level during S-phase, whereas the levels of RRM1 remain relatively constant throughout the cell cycle. Fluctuations in RRM2 protein levels have been attributed to both transcriptional up-regulation during S-phase and proteasome-mediated degradation as cells enter mitosis (11.Nordlund P. Reichard P. Annu. Rev. Biochem. 2006; 75: 681-706Crossref PubMed Scopus (842) Google Scholar). DNA damage also regulates RNR activity. In budding yeast RNR up-regulation (14.Lubelsky Y. Reuven N. Shaul Y. Mol. Cell. Biol. 2005; 25: 10665-10673Crossref PubMed Scopus (39) Google Scholar, 15.Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (294) Google Scholar) depends on the protein kinases Mec1 and Rad53. Mec1 initiates Rad53 activation by phosphorylating Rad53, and Rad53 is further activated by autophosphorylation. Activated Rad53 up-regulates RNR by phosphorylating Dun1, another protein kinase (16.Chen S.H. Smolka M.B. Zhou H. J. Biol. Chem. 2007; 282: 986-995Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Activated (phosphorylated) Dun1 up-regulates RNR by at least two routes (see Fig. 7). The first is through phosphorylation of Sml1 (17.Zhao X. Muller E.G. Rothstein R. Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 18.Zhao X. Chabes A. Domkin V. Thelander L. Rothstein R. EMBO J. 2001; 20: 3544-3553Crossref PubMed Scopus (227) Google Scholar), which dissociates Sml1 from RNR and de-represses the activity of RNR (19.Uchiki T. Dice L.T. Hettich R.L. Dealwis C. J. Biol. Chem. 2004; 279: 11293-11303Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 20.Zhao X. Rothstein R. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 3746-3751Crossref PubMed Scopus (217) Google Scholar). The second route by which Dun1 up-regulates RNR is through phosphorylation of Crt1, which dissociates Crt1 from the RNR promoter and de-represses RNR gene transcription (21.Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Thus, in budding yeast, the DNA damage response kinases Mec1-Rad53-Dun1 act as positive regulators of RNR both at the transcriptional and post-transcriptional levels. In mammalian cells evidence is limited regarding the regulation of RNR by the orthologs of the yeast Mec1 and Rad53, ATM/ATR and Chk1/Chk2, respectively (22.Elledge S.J. Zhou Z. Allen J.B. Navas T.A. BioEssays. 1993; 15: 333-339Crossref PubMed Scopus (210) Google Scholar, 23.Naruyama H. Shimada M. Niida H. Zineldeen D.H. Hashimoto Y. Kohri K. Nakanishi M. Biochem. Biophys. Res. Commun. 2008; 374: 79-83Crossref PubMed Scopus (21) Google Scholar). Nevertheless, the mammalian Crt1 ortholog, Rfx1, has been found to bind to the RNR2 gene and block its transcription (14.Lubelsky Y. Reuven N. Shaul Y. Mol. Cell. Biol. 2005; 25: 10665-10673Crossref PubMed Scopus (39) Google Scholar). Meanwhile, RNR genes contain E2F binding sites and could be activated at the transcription level by E2F1 overexpression in quiescent cells before the induction of S-phase (13.DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (843) Google Scholar, 24.Chabes A.L. Björklund S. Thelander L. J. Biol. Chem. 2004; 279: 10796-10807Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). E2F1 activity is also up-regulated in response to CPT (25.Fusaro G. Wang S. Chellappan S. Oncogene. 2002; 21: 4539-4548Crossref PubMed Scopus (95) Google Scholar). A recent study also provided evidence for RNR2 regulation by Chk1 for S-phase progression (23.Naruyama H. Shimada M. Niida H. Zineldeen D.H. Hashimoto Y. Kohri K. Nakanishi M. Biochem. Biophys. Res. Commun. 2008; 374: 79-83Crossref PubMed Scopus (21) Google Scholar). Ataxia telangiectasia (AT) cells are also deficient in activating p53R2 compared with normal wild type cells (26.Eaton J.S. Lin Z.P. Sartorelli A.C. Bonawitz N.D. Shadel G.S. J. Clin. Invest. 2007; 117: 2723-2734Crossref PubMed Scopus (150) Google Scholar, 27.Yamaguchi T. Matsuda K. Sagiya Y. Iwadate M. Fujino M.A. Nakamura Y. Arakawa H. Cancer Res. 2001; 61: 8256-8262PubMed Google Scholar). Our present findings indicate that up-regulation of RRM2 transcription in response to DNA damage in human cells involves an ATR/ATM-Chk1-E2F1 pathway, which is reminiscent of the Mec1-Rad53-Dun1 pathway in budding yeast (see Fig. 7). MDA-MB-231 breast cancer cells and HCT-116 colorectal cancer cells were obtained from the Developmental Therapeutics Program (NCI, National Institutes of Health, nci.nih.gov) and were maintained in RPMI 1640 medium containing 10% fetal bovine serum. All siRNAs were obtained from Qiagen Inc. (Germantown, MD). CPT was obtained from Sigma. UCN-01 was obtained from the Developmental Therapeutics Program. CHIR124 was a kind gift from Chiron Corp. KU-55933 was a kind gift from KuDOS Pharmaceuticals Ltd. (Cambridge, UK). CPT and KU-55933 were prepared at 10 mm in DMSO. UCN-01 and CHIR124 were prepared at 1 mm in DMSO. Drug stock solutions were separated into aliquots at −20 °C. All drugs were diluted to desired concentrations in full medium immediately before each experiment. The final DMSO concentrations did not exceed 0.1%. The CPT chemosensitization RNAi screen was performed using a library of synthetic siRNAs targeting ≈400 genes associated with cancer and using a previously described multiplexed siRNA screening strategy (see Martin et al. (28.Martin S.E. Jones T.L. Thomas C.L. Lorenzi P.L. Nguyen D.A. Runfola T. Gunsior M. Weinstein J.N. Goldsmith P.K. Lader E. Huppi K. Caplen N.J. Nucleic Acids Res. 2007; 35: e57Crossref PubMed Scopus (21) Google Scholar) for details). Multiplexes comprised six siRNAs corresponding to three unique gene targets (two siRNAs per gene). Multiplexes were evaluated at a final concentration of 60 nm (10 nm of each individual siRNA) in a 96-well-plate format. Only the interior 60 wells were used. Transfections were performed by precomplexing siRNA (6 pmol) with Oligofectamine lipid transfection reagent (Invitrogen) in 50 μl of serum-free RPMI in individual plate wells for 30 min at ambient temperature. MDA-MB-231 cells (4500) were added in 50 μl of RPMI supplemented with 10% fetal bovine serum to yield transfection mixtures consisting of 60 nm total siRNA in RPMI with 5% fetal bovine serum. This final mixture was incubated at ambient temperature for 45 min before being placed at 37 °C in a humidified atmosphere containing 5% CO2. The library was screened in duplicate (half intended to receive CPT treatment and half to establish siRNA multiplex basal activity). After 48 h the medium was removed, and 100 μl of fresh medium containing either CPT (≈EC50, 0.1% DMSO) or vehicle only (0.1% DMSO) was added, and the cells incubated for an additional 48 h at 37 °C. After this time, cell viability was assayed (Cell Titer Blue Reagent, Promega, Madison, WI). Plate median values were used for normalization. Multiplexes were assayed in duplicate. For deconvolution studies, the two siRNAs targeting a given gene were evaluated as a pair, each used at 10 nm, and compared with cells transfected with negative control siRNA (siNeg) (20 nm). For RNA analysis, transfections were performed as described for screening, except that 2500 cells were seeded. Unless otherwise stated, after 48 h the medium was removed, 100 μl of fresh medium containing CPT (1 μm, 0.1% DMSO) was added, and the cells were incubated at 37 °C for 24–72 h depending on the end assay. For protein analysis, transfections were performed in 6-well plates with Oligofectamine reagent. Cells transfected with negative control siRNA were used for comparison. The target sequences for the experimental siRNAs are listed in supplemental Table 1. After 30 min of pulse-labeling with 50 μmol/liter bromodeoxyuridine (BrdUrd, Calbiochem), cells were collected and stained with fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (BD Biosciences). Cells were resuspended in 500 μl of propidium iodide (PI) solution (50 μg/ml PI and 50 μg/ml RNase A) and analyzed with a FACScan flow cytometer (BD Biosciences). Gene-specific transcript levels were measured using a branched DNA-based assay (QuantiGene Reagent System, Panomics, Fremont, CA) in single or multiplex formats. The single gene assay format has been described previously (28.Martin S.E. Jones T.L. Thomas C.L. Lorenzi P.L. Nguyen D.A. Runfola T. Gunsior M. Weinstein J.N. Goldsmith P.K. Lader E. Huppi K. Caplen N.J. Nucleic Acids Res. 2007; 35: e57Crossref PubMed Scopus (21) Google Scholar). In this study probes corresponding to RRM1, RRM2, or CTNNB1 (control) and human cyclophilin (PPIB) (for normalization) (Panomics) were used. The multiplex format (Quantigene® Plex assay, Panomics) was performed using a custom-designed panel consisting of 20 different XMAP® beads, each conjugated with a probe set corresponding to a different mRNA. The mRNAs assayed included genes associated with this study (RRM2, RRM1, E2F1, CHEK1, CHEK2, ATM, and ATR). Bead identity was used to identify each mRNA species, and the fluorescent signal from each bead (Bioplex, Bio-Rad) was used to quantify the amount of RNA. Protein levels were measured by Western blot with corresponding specific primary antibodies, including those against RRM2, Chk1, and E2F1 (Santa Cruz, CA), phosphorylated-H2AX (γ-H2AX) (Upstate/Millipore), RRM1 (Chemicon/Millipore), glyceraldehyde-3-phosphate dehydrogenase, and phosphorylated-Chk1(Ser-317) (Cell Signaling). Shown are the representative data from separate experiments. MDA-MB-231 cells plated in four-well chamber slides (Nalgene Nunc International, Rochester, NY) were used to study the subcellular localization of RRM2. After drug treatment, cells were fixed for 20 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) and washed twice with PBS. After incubation for 20 min with 70% ethanol and washing with PBS, the cells were incubated in blocking buffer (8% bovine serum albumin in PBS) for 1 h before incubation for 2 h with primary antibodies against RRM2 (Santa Cruz, CA). Slides were incubated for an additional 1 h with the Alex488-conjugated secondary antibody (Alexa Fluor® 488 donkey anti-goat IgG, Molecular Probes/Invitrogen). After three washes in PBS, cells were stained with 0.5 μg/ml PI and 100 μg/ml RNaseA (Sigma) for 15 min in the dark. Finally, slides were washed with PBS for three times and mounted with Vectashield anti-fade mounting media (Vector Laboratories, Inc., Burlingame, CA). Images were taken using a Nikon Eclipse TE-300 confocal microscope. All the data are represented as mean values ± S.D. The significance of differences between means was assessed by the Student's t test, with p < 0.05 being considered statistically significant. Early studies of CPT showed this compound was cytotoxic to breast cancer cell lines (29.Nieves-Neira W. Pommier Y. Int. J. Cancer. 1999; 82: 396-404Crossref PubMed Scopus (119) Google Scholar); however, adverse in vivo side effects and/or drug resistance due to overexpression of ABCG2 (30.Brangi M. Litman T. Ciotti M. Nishiyama K. Kohlhagen G. Takimoto C. Robey R. Pommier Y. Fojo T. Bates S.E. Cancer Res. 1999; 59: 5938-5946PubMed Google Scholar, 31.Doyle L.A. Ross D.D. Oncogene. 2003; 22: 7340-7358Crossref PubMed Scopus (931) Google Scholar) have limited the clinical development of the CPT derivatives as main line treatments for breast cancer. Recent studies have, however, reconsidered the potential of CPT in treatment of advanced breast cancer alone and in combination with other chemotherapeutic agents (32.O'Connor T. Rustum Y. Levine E. Creaven P. Cancer Chemother. Pharmacol. 2008; 61: 125-131Crossref PubMed Scopus (9) Google Scholar). A rational approach to determining additional pathways that could be used to improve the application of Top1 inhibitors in advanced breast cancer could be revealed by RNAi screening in an appropriate model system. The breast cancer MDA-MB-231 cell line is frequently used as a cellular model of triple-negative, basal-like breast cancer as it lacks expression of the estrogen receptor, progesterone receptor, and ERBB2 (HER2/Neu). A multiplex siRNA-based RNAi screen (28.Martin S.E. Jones T.L. Thomas C.L. Lorenzi P.L. Nguyen D.A. Runfola T. Gunsior M. Weinstein J.N. Goldsmith P.K. Lader E. Huppi K. Caplen N.J. Nucleic Acids Res. 2007; 35: e57Crossref PubMed Scopus (21) Google Scholar) was performed in the MDA-MB-231 to identify genes that modulated the activity of CPT (Fig. 1A). A duplicate screen showed good correlation (r = 0.8, data not shown). Deconvolution of the top sensitizing siRNA multiplexes identified RRM1 and RRM2 as genes whose silencing not only affected cell viability alone but also significantly enhanced the cytotoxicity of CPT (multiplexes C and G, Fig. 1B). As expected (33.Pommier Y. Barcelo J.M. Rao V.A. Sordet O. Jobson A.G. Thibaut L. Miao Z.H. Seiler J.A. Zhang H. Marchand C. Agama K. Nitiss J.L. Redon C. Prog. Nucleic Acid Res. Mol. Biol. 2006; 81: 179-229Crossref PubMed Scopus (231) Google Scholar), BRCA1 and ATR knockdowns also produced CPT sensitization (multiplexes E and F, respectively). This screen also identified gap junction protein β1 (GJB1), a member of the connexin family of proteins, as an additional gene whose silencing enhances CPT toxicity. Notably, connexin proteins have been linked to mechanisms of cell death (34.Decrock E. Vinken M. De Vuyst E. Krysko D.V. D'Herde K. Vanhaecke T. Vandenabeele P. Rogiers V. Leybaert L. Cell Death Differ. 2009; 16: 524-536Crossref PubMed Scopus (213) Google Scholar). However, this target was not pursued further in the context of this study. HU inactivates RNR by targeting specifically the RRM2 subunit (35.Lassmann G. Thelander L. Gräslund A. Biochem. Biophys. Res. Commun. 1992; 188: 879-887Crossref PubMed Scopus (86) Google Scholar). In contrast to RRM2 knockdown (Fig. 1B), HU is known to protect from the cytotoxicity of CPT (7.Holm C. Covey J.M. Kerrigan D. Pommier Y. Cancer Res. 1989; 49: 6365-6368PubMed Google Scholar, 36.Cheng M.F. Chatterjee S. Berger N.A. Oncol. Res. 1994; 6: 269-279PubMed Google Scholar). To clarify the difference between the effects of HU and RRM2 silencing on CPT cytotoxicity, a BrdUrd incorporation assay was used to detect the DNA replication in cells treated with HU or with RRM2 siRNA. Five mm HU blocked DNA replication totally after 24 h of treatment (Fig. 2A). On the other hand, although the RRM2 protein level was knocked down with siRNA (Fig. 2B), only limited effects on cell cycle distribution were produced. siRNA against RRM2 produced an increase of S-phase cells but without fully arresting DNA synthesis as most cells in S-phase continued to incorporate BrdUrd (Fig. 2C). Thus, RRM2 knockdown, unlike HU, only partially reduced DNA synthesis. Because in yeast cells, the two RNR genes encoding the small (RNR2) and large (RNR3) subunits are both up-regulated at the transcriptional level in response to DNA damage (15.Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (294) Google Scholar), we hypothesized that CPT could also up-regulate the mammalian RNR genes, which may form part of the basis for the sensitization to CPT seen after RNAi against the RNR genes. To assess this possibility we examined the protein and mRNA levels of RRM1 and RRM2 after treatment of MDA-MB-231 cells with CPT. At the protein level both RRM2 and RRM1 were induced, and RRM2 showed the greatest increase after CPT addition (Fig. 3A). At the mRNA level, RRM2 expression was induced ∼2.5-fold, and RRM1 was induced about 1.8-fold, 24–48 h post-CPT addition (Fig. 3B). RRM1 and RRM2 induction was also observed in the human colon carcinoma cell line HCT-116 (Fig. 3C). Because the RRM2 induction after CPT treatment was more pronounced than the RRM1 induction, and because of the regulatory role of the RNR2 subunit in the DNA damage response of yeast, we choose to focus our subsequent studies on RRM2. Immunofluorescence assays were performed to further examine the RRM2 induction by CPT at different time points up to 24 h. The overall RRM2 fluorescence signal per cell rose with drug exposure time, and RRM2 induction was already detectable after 1 h of CPT treatment (Fig. 3D). Moreover, subcellular localization analyses showed translocation of RRM2 from cytoplasm to nucleus in CPT-treated cells. Compared with the untreated control, the population of cells with nuclear RRM2 signal after CPT treatment increased from 7 to 37% (Fig. 3E). Those data confirmed the induction of RRM2 expression in response to CPT and revealed nuclear translocation of RRM2, consistent with the potential role of RRM2 in the DNA repair of Top1-induced DNA damage. To probe the DNA damage response mechanisms underlying the transcriptional up-regulation of RRM2 induced by CPT, we investigated the effects of RNAi targeting RRM2, E2F1, CHEK1, CHEK2, ATM, and ATR on the expression of those genes in the absence and presence of CPT. Each siRNA showed high efficiency silencing of the corresponding target gene expression in the absence (and presence) of CPT. As seen in Fig. 4, besides RRM2, silencing of CHEK1, E2F1, ATM, or ATR all partially suppressed the up-regulation of RRM2 by CPT. However, CHEK2 silencing had no effect on the RRM2 induction. The silencing of CHEK1 also suppressed the up-regulation of E2F1 expression, and silencing of ATM and ATR blocked both CHEK1 and E2F1 up-regulation at transcription level. Together these results suggested that ATM and ATR regulate RMM2 via CHEK1 and E2F1. In yeast cells, Rad53 regulates the expression of the ribonucleotide reductase small subunit RNR2 in response to DNA damage (37.Allen J.B. Zhou Z. Siede W. Friedberg E.C. Elledge S.J. Genes Dev. 1994; 8: 2401-2415Crossref PubMed Scopus (344) Google Scholar, 38.Koc A. Merrill G.F. Biochem. Biophys. Res. Commun. 2007; 353: 527-530Crossref PubMed Scopus (8) Google Scholar, 39.Woolstencroft R.N. Beilharz T.H. Cook M.A. Preiss T. Durocher D. Tyers M. J. Cell Sci. 2006; 119: 5178-5192Crossref PubMed Scopus (50) Google Scholar), but in mammalian cells the regulation of RRM2 is still unclear. Of the mammalian homologues of Rad53, Chk1, and Chk2, only the silencing of CHEK1 suppressed the transcription up-regulation of RRM2 in response to CPT, whereas CHEK2 silencing had no effect (Fig. 4). Therefore, we investigated the relationship between Chk1 and RRM2 in MDA-MB-231 cells treated with CPT. Fig. 5 shows that Chk1 was activated by CPT. Indeed, phosphorylated-Chk1 (Chk1-Ser-317) increased upon CPT treatment (Fig. 5A). Both the Chk1 inhibitors UCN-01 and CHIR124 decreased the up-regulation of RRM2 in response to CPT, which suggested that Chk1 was involved in the up-regulation of RRM2 in response to CPT treatment (Fig. 5B). This was further confirmed by knockdown experiments with siRNA, which showed an attenuation of the up-regulation of RRM2 by CHEK1 knockdown (Fig. 5C). CHEK1 knockdown also enhanced the γ-H2AX response to CPT. Taken together, those experiments demonstrate that the up-regulation of RRM2 induced by CPT is Chk1-dependent and may have a protective effect as inactivation of Chk1 enhances CPT-induced DNA damage (9.Furuta T. Takemura H. Liao Z.Y. Aune G.J. Redon C. Sedelnikova O.A. Pilch D.R. Rogakou E.P. Celeste A. Chen H.T. Nussenzweig A. Aladjem M.I. Bonner W.M. Pommier Y. J. Biol. Chem. 2003; 278: 20303-20312Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 40.Furuta T. Hayward R.L. Meng L.H. Takemura H. Aune G.J. Bonner W.M. Aladjem M.I. Kohn K.W. Pommier Y. Oncogene. 2006; 25: 2839-2849Crossref PubMed Scopus (42) Google Scholar). Because the RNR gene is one of the transcriptional targets of the replication- and stress-associated transcription factor E2F1 (13.DeGregori J. Kowalik T. Nevins J.R. Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (843) Google Scholar, 24.Chabes A.L. Björklund S. Thelander L. J. Biol. Chem. 2004; 279: 10796-10807Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and because E2F1 has been involved in the DNA damage response through its phosphorylation by ATM and Chk2 (41.Lin W.C. Lin F.T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar, 42.Stevens C. Smith L. La Thangue N.B. Nat. Cell Biol. 2003; 5: 401-409Crossref PubMed Scopus (327) Google Scholar), we investigated in more details the role of E2F1 in the up-regulation of RRM2 in response to CPT. After our finding that E2F1 increased at the mRNA level under exposure to CPT (Fig. 4), we examined E2F1 response at the protein level. Fig. 5 (B–D) shows enhanced E2F1 protein signal in cells treated with CPT. To determine the effect of E2F1 on the RRM2 up-regulation, knockdown of E2F1 with siRNA was then introduced. We had already found that E2F1 silencing suppressed RRM2 transcriptional up-regulation in response to CPT (Fig. 4). E2F1 knockdown also blocked the up-regulation of RRM2 at the protein level (Fig. 5D). These data demonstrate that RRM2 up-regulation in response to CPT is regulated by E2F1. The next set of experiments was done to examine the relationship between Chk1 and E2F1 in the RRM2 up-regulation. After siChk1 silencing, E2F1 base-line expression was reduced at the protein leve
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