Stable Suppression of the R2 Subunit of Ribonucleotide Reductase by R2-targeted Short Interference RNA Sensitizes p53(–/–) HCT-116 Colon Cancer Cells to DNA-damaging Agents and Ribonucleotide Reductase Inhibitors

Abstract

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(–/–) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(–/–) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine® and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(–/–) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2. Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(–/–) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(–/–) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine® and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(–/–) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2. Ribonucleotide reductase (RNR) 1The abbreviations used are: RNR, ribonucleotide reductase; R2-RNR, RNR containing the R2 subunit; p53R2-RNR, RNR containing the p53R2 subunit; sh/siRNA, short hairpin/short interference RNA; CMV, cytomegalovirus; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl) -2H-tetrazolium.1The abbreviations used are: RNR, ribonucleotide reductase; R2-RNR, RNR containing the R2 subunit; p53R2-RNR, RNR containing the p53R2 subunit; sh/siRNA, short hairpin/short interference RNA; CMV, cytomegalovirus; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl) -2H-tetrazolium. catalyzes a rate-limiting reaction in which ribonucleoside diphosphates are converted to their corresponding deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis and repair (1Thelander L. Reichard P. Annu. Rev. Biochem. 1979; 48: 133-158Crossref PubMed Scopus (909) Google Scholar). In mammalian cells, the catalytically active RNR is considered to be an α2β2 heterotetramer consisting of two large R1 subunits and two small R2 subunits. To maintain a balanced size of dNTP pools (2Kunz B.A. Kohalmi S.E. Kunkel T.A. Mathews C.K. McIntosh E.M. Reidy J.A. Mutat. Res. 1994; 318: 1-64Crossref PubMed Scopus (78) Google Scholar), the enzymatic activity of RNR is tightly regulated by the binding of nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) to allosteric sites in the R1 subunit (1Thelander L. Reichard P. Annu. Rev. Biochem. 1979; 48: 133-158Crossref PubMed Scopus (909) Google Scholar). In proliferating cells, the level of the R2 protein is low in the G1 phase of the cell cycle but accumulates and reaches maximal levels during the S phase, followed by degradation when passing through the G2/M phase (3Eriksson S. Graslund A. Skog S. Thelander L. Tribukait B. J. Biol. Chem. 1984; 259: 11695-11700Abstract Full Text PDF PubMed Google Scholar, 4Engstrom Y. Eriksson S. Jildevik I. Skog S. Thelander L. Tribukait B. J. Biol. Chem. 1985; 260: 9114-9116Abstract Full Text PDF PubMed Google Scholar, 5Bjorklund S. Skog S. Tribukait B. Thelander L. Biochemistry. 1990; 29: 5452-5458Crossref PubMed Scopus (157) Google Scholar, 6Mann G.J. Musgrove E.A. Fox R.M. Thelander L. Cancer Res. 1988; 48: 5151-5156PubMed Google Scholar). Thus, the activity of RNR is additionally controlled by cell cycle-specific availability of the R2 subunit, whereas the R1 protein level remains relatively constant throughout the cell cycle (3Eriksson S. Graslund A. Skog S. Thelander L. Tribukait B. J. Biol. Chem. 1984; 259: 11695-11700Abstract Full Text PDF PubMed Google Scholar, 4Engstrom Y. Eriksson S. Jildevik I. Skog S. Thelander L. Tribukait B. J. Biol. Chem. 1985; 260: 9114-9116Abstract Full Text PDF PubMed Google Scholar). When cells undergo G1 arrest following DNA damage, the supply of dNTPs for DNA repair is, in turn, provided by transcriptional activation of the recently identified R2 homologue, p53R2 (7Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (741) Google Scholar, 8Nakano K. Balint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar). Thus, DNA damage causes p53-dependent induction of p53R2 that complexes with R1 to form active RNR in arrested cells (9Guittet O. Hakansson P. Voevodskaya N. Fridd S. Graslund A. Arakawa H. Nakamura Y. Thelander L. J. Biol. Chem. 2001; 276: 40647-40651Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The discovery of p53R2 by Tanaka et al. (7Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (741) Google Scholar) provides an explanation of how cells with damaged DNA can acquire a supply of dNTPs for DNA repair when the R2 level is repressed during G1 arrest; they proposed two independent pathways that lead to the supply of dNTPs in mammalian cells. One is mediated by the R2 subunit of RNR in maintaining dNTP pools for DNA replication during the S phase of the cell cycle. The other involves p53R2 to supply dNTPs for DNA repair when DNA damage occurs (7Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (741) Google Scholar, 10Yamaguchi T. Matsuda K. Sagiya Y. Iwadate M. Fujino M.A. Nakamura Y. Arakawa H. Cancer Res. 2001; 61: 8256-8262PubMed Google Scholar). However, with >50% of human cancers containing mutated or deleted p53, it is unclear whether malignant cells that lack inducible p53R2 are capable of supplying sufficient dNTPs through this pathway for DNA repair. Early studies have demonstrated a positive correlation between the enzymatic activity of RNR and the rate of proliferation of transplanted Novikoff carcinomas (11Elford H.L. Freese M. Passamani E. Morris H.P. J. Biol. Chem. 1970; 245: 5228-5233Abstract Full Text PDF PubMed Google Scholar, 12Weber G. Oncology. 1980; 37: 19-24Crossref PubMed Scopus (5) Google Scholar). Thus, because of its critical role in the de novo synthesis of DNA, RNR is considered to be a potential target for the development of cancer chemotherapeutic agents. At present, hydroxyurea is the only clinically used RNR inhibitor; however, several more potent and specific inhibitors of RNR have been developed and are currently being evaluated clinically (13Cory J.G. Cory A.H. Rappa G. Lorico A. Liu M.C. Lin T.S. Sartorelli A.C. Biochem. Pharmacol. 1994; 48: 335-344Crossref PubMed Scopus (114) Google Scholar, 14Finch R.A. Liu M.C. Cory A.H. Cory J.G. Sartorelli A.C. Adv. Enzyme Regul. 1999; 39: 3-12Crossref PubMed Scopus (216) Google Scholar, 15Fritzer-Szekeres M. Salamon A. Grusch M. Horvath Z. Hochtl T. Steinbrugger R. Jager W. Krupitza G. Elford H.L. Szekeres T. Biochem. Pharmacol. 2002; 64: 481-485Crossref PubMed Scopus (24) Google Scholar, 16Inayat M.S. Chendil D. Mohiuddin M. Elford H.L. Gallicchio V.S. Ahmed M.M. Cancer Biol. Ther. 2002; 1: 539-545Crossref PubMed Scopus (32) Google Scholar, 17Liu M.C. Lin T.S. Sartorelli A.C. J. Med. Chem. 1992; 35: 3672-3677Crossref PubMed Scopus (156) Google Scholar). We have shown previously that the RNR inhibitor Triapine, in conjunction with a variety of DNA-damaging agents, produces synergistic inhibition of transplanted L1210 leukemia cells, presumably by inhibiting the ability of cancer cells to repair drug-induced damage to DNA (18Finch R.A. Liu M. Grill S.P. Rose W.C. Loomis R. Vasquez K.M. Cheng Y. Sartorelli A.C. Biochem. Pharmacol. 2000; 59: 983-991Crossref PubMed Scopus (342) Google Scholar). Thus, it is important to fully understand the regulation of RNR and its role in DNA repair, for cancer cells with mutated In the have the regulation of the R2 and p53R2 subunits of RNR in response to DNA damage in p53(+/+) and p53(–/–) HCT-116 human colon cancer cell the of RNA have p53(–/–) HCT-116 cell stably R2-targeted that causes of an of the role of the R2 subunit in the sensitivity of cells to DNA damage and to the inhibition of The findings that the R2 subunit of RNR can for the of p53R2 in dNTPs for DNA repair in cells and and p53(–/–) human colon cancer cells were provided by were in with and p53R2, and were and were vector and containing p53R2 K. Balint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar) were Vousden of The the S M. A. Yamaguchi I. 1994; PubMed Scopus Google Scholar) and the the P. A. M. G. 1988; PubMed Scopus Google Scholar) were G. D. D. R. J. 1988; PubMed Scopus Google Scholar) containing the was provided by The DNA of the and the to R2-targeted were by the as a The a of human R2 by a the to the and followed by as the transcriptional The used are the and the or the were the vector The and of the were by and DNA The were the vector by with and To the R2 expression vector or the the was which the was by with and HCT-116 cells were in for to were with of the vector or with of the to the cells were and in a containing S. for of cells were and with a growth containing S. cells were for an and stable cell stably were and identified by and stable of stable p53(–/–) cell were transfected with of the vector or as cells were and in a containing of cells were and cell The cell were of stable cell used in the studies were cells in of their expression and were with and and through a to were by the protein to the of protein were by a and a The was with in and for and with in the The was with with a in for and The target protein was by the enhanced cells were in and with inhibitor The was and with for and with protein with constant The were and in and and for The the was for by were in and for was to in a of of of One was and the was for an The of by cells following was a The sensitivity of cells to was as a of the of dNTP deoxyribonucleoside triphosphates were as by and J.A. Biochem. PubMed Scopus Google Scholar) and by et al. H. Biochem. 1994; PubMed Scopus Google Scholar). cells were or with cisplatin or Triapine, with and with The were to for to cell and The following were used in the for for for dGTP for dATP and a The reaction of DNA and for and dGTP or for dATP of cells was to of the reaction and for of the was which was with with and with and The the was by The of DNA was to the of the dNTP being DNA by and p53R2 and R2 in p53(+/+) and p53(–/–) HCT-116 induction of p53R2 in response to DNA damage been shown to (7Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (741) Google Scholar, 8Nakano K. Balint E. Ashcroft M. Vousden K.H. Oncogene. 2000; 19: 4283-4289Crossref PubMed Scopus (199) Google Scholar). To the of in the expression of p53R2 and R2 following DNA damage, protein levels of subunits in p53(+/+) and p53(–/–) HCT-116 human colon cancer cells were evaluated by shown in with a potent DNA-damaging caused a and induction of p53R2 in p53(+/+) but not in p53(–/–) cells. The other protein K. Y. B. Nature. PubMed Scopus Google Scholar), exhibited a response to The R2 subunit an expression that of p53R2. Thus, to doxorubicin for resulted in the of R2 p53(+/+) cells, presumably by degradation In of p53(–/–) cells with doxorubicin caused a small but steady increase in R2 of p53(+/+) cells with cisplatin resulted in a of R2 degradation to that caused by doxorubicin The of the R2 protein caused by cisplatin relatively These results that R2 protein levels elevated by DNA damage in p53(–/–) cells in which p53-dependent p53R2 induction and R2 degradation pathways are of p53(+/+) and p53(–/–) HCT-116 to and sensitivity of p53(+/+) and p53(–/–) HCT-116 cells to of the DNA-damaging chemotherapeutic and cisplatin were with a of cytotoxicity by the an agent that P. J. L. Cancer Res. Google Scholar, Oncogene. 2001; PubMed Scopus Google Scholar) but not damage DNA, was in the demonstrated in p53(+/+) cells exhibited sensitivity to and p53(–/–) HCT-116 cells. Thus, a lack of in p53(–/–) cells resulted in a in the sensitivity to the DNA-damaging and agents. that the level of p53R2 in response to DNA damage not be in p53(+/+) HCT-116 cells or not sufficient levels of dNTPs for DNA repair. a in sensitivity to inhibition by was it is that activation by the employed cell cycle arrest that the repair of Ectopic of p53R2 in p53(–/–) HCT-116 the role of p53R2 in DNA repair and sensitivity to DNA-damaging agents, it was important to the of other p53-dependent as the survival of HCT-116 cells. To this stable p53(–/–) HCT-116 cell transfected with the p53R2 expression vector and for expression of p53R2 were of an vector as and p53R2 expression stable clones are shown in relatively of p53R2 was in clones and the of this expression was an the exhibited a low level of p53R2. The levels of p53R2 protein in p53(–/–) HCT-116 cells were with of the p53R2 in p53(+/+) HCT-116 cells Furthermore, expression of p53R2 not in the levels of that the expression of is regulated in the of DNA of and DNA of R2 in p53(–/–) HCT-116 by of the R2 studies p53(–/–) HCT-116 cell that or no change in RNR activity or sensitivity to DNA-damaging not We that the of p53R2 to RNR activity be by and levels of R2 in cells. to the levels of R2 in p53(–/–) HCT-116 cells, a expression vector transcribing an directed against the human R2 The as well as and cells were stably transfected with either the vector or the expression to stable transfected clones were and cell and for suppression of R2 levels by of clones a >50% reduction in R2 protein levels not corresponding to or an reduction in the R2 was and is shown in The knockdown of R2 not p53R2 and clones exhibited no in growth rate as with their that have levels of the R2 To whether in the of RNR subunits in cells, were protein and their corresponding clones was with an were by and shown in p53R2 with R1 was only in but not in cells. that p53R2 in p53(–/–) cells RNR with an R1 R2 with R1 was in and cells. of R2 in and cells caused a in the levels of R2 with is with a in the level of R2 in cells by DNA damage caused by doxorubicin R2 in p53(–/–) HCT-116 cells whether stable knockdown of R2 the induction of R2 by with DNA-damaging agents. and as well as their corresponding were to or for and the expression of R2 protein was by that with cisplatin and but not with resulted in the induction of R2 in and cells. DNA damage by cisplatin and doxorubicin caused the induction of R2 in but the induced levels of R2 were that in R2 cells. of p53(–/–) HCT-116 to the DNA-damaging and the RNR and by of the R2 of R2 knockdown and p53R2 expression the sensitivity of p53(–/–) HCT-116 cells to DNA damage and RNR inhibition was evaluated in and their corresponding cells. to of Triapine, and for cytotoxicity was by shown in suppression of R2 levels by stable cells to In and cells with levels of the R2 subunit exhibited only a increase in sensitivity to the knockdown of and cells exhibited but more to cisplatin cells. The of sensitivity to doxorubicin was to that of the sensitivity to cisplatin with cells not These results that the sensitivity of p53(–/–) HCT-116 cells to DNA damage caused by cisplatin and doxorubicin is by the levels of However, R2 only to DNA damage in cells p53R2. knockdown of R2 in and cells caused a pronounced increase in sensitivity to the RNR inhibitors and hydroxyurea and In this clones with levels of R2 exhibited enhanced sensitivity to agents. Thus, expression of p53R2 in clones and cells not the of the RNR inhibitors when the level of R2 is These findings that the level of R2 the sensitivity of p53(–/–) HCT-116 cells to the inhibitors of In expression of p53R2 not to for the levels of R2 caused by stable and their corresponding clones in their sensitivity to the agent of DNA of dNTPs by of R2-targeted in p53(–/–) HCT-116 in dNTP pools with R2 and p53R2 expression were in and and their corresponding cells with The levels of dNTP were in cells to cisplatin for a and which induction of the R2 protein was demonstrated in with cisplatin caused a marked increase in the levels of dNTPs in of the cell R2 knockdown resulted in of elevated dNTP levels in cells. In the levels of dNTPs were relatively by R2 in cells with These results that R2 knockdown to the suppression of the DNA elevation of dNTP levels in cells. Ectopic expression of p53R2 in cells be capable of partially the in dNTP levels caused by R2 of dATP and dGTP in p53(–/–) HCT-116 by the of RNR by RNR inhibitor cytotoxicity cells R2 cells of the of expression of p53R2 To whether this sensitivity correlated with in RNR the levels of the dNTPs were in the and of and and their corresponding cells were to for this resulted in a pronounced in dATP levels and a reduction in dGTP In the levels of and were by in of the cell Furthermore, R2 in and cells to a reduction in the levels of dATP and that in cells R2 and in the of the of R2 dGTP levels were relatively small These results that the knockdown of R2 the of the suppression of dATP cells exhibited a of in dATP and dGTP levels to that in cells in response to is with the cytotoxicity that expression of p53R2 for the in dATP pools caused by the knockdown of the R2 subunit following with The relatively discovery of p53R2 not only provides evidence of the involvement of in DNA repair but a the the regulation of RNR in the synthesis of DNA and the response to DNA results that R2 as p53R2 following drug-induced damage to DNA in p53(+/+) HCT-116 cells. was with other cell (7Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (741) Google Scholar, 10Yamaguchi T. Matsuda K. Sagiya Y. Iwadate M. Fujino M.A. Nakamura Y. Arakawa H. Cancer Res. 2001; 61: 8256-8262PubMed Google Scholar). These findings during DNA G1 p53R2 R2 in an active RNR with the R1 subunit in a p53-dependent Thus, it is that cells with are in damaged DNA and, to DNA-damaging agents. is by studies that of of cells to DNA damage by cancer chemotherapeutic S. D. Cancer Res. Google Scholar, Cancer Res. Google Scholar). In however, that p53(+/+) HCT-116 cells sensitivity to DNA-damaging and p53(–/–) cells. Yamaguchi et al. T. Matsuda K. Sagiya Y. Iwadate M. Fujino M.A. Nakamura Y. Arakawa H. Cancer Res. 2001; 61: 8256-8262PubMed Google Scholar) that HCT-116 cells a in the p53R2 subunit results in a of the by and a of RNR activity in cells. that a in the of p53R2 lead to the activation of the pathway in HCT-116 cells. Thus, p53-dependent or DNA repair, it is that a lack of in cells a in sensitivity to chemotherapeutic DNA The involvement of the R2 subunit in DNA repair remains have shown that DNA damage the expression of the of mammalian and that of the results in sensitivity to DNA damage Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). is to that R2 is induced by DNA damage in mammalian cells R.A. J.A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, D. S. E. Thelander L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Cancer Res. Google Scholar). However, the of the mammalian R2 subunit to DNA repair is In cells, R2 is tightly regulated in a an level of the R2 protein occurs during the S phase or during a which been to an of the protein but not to the elevated of the A. Thelander L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Thelander L. S. A. PubMed Scopus Google Scholar). of cells the R2 protein is by the presumably to DNA synthesis A. Thelander L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Thelander L. S. A. PubMed Scopus Google Scholar). These results findings in p53(+/+) HCT-116 cells, because cells R2 degradation following and of to doxorubicin and as a of the cells p53-dependent G1 arrest passing through the G2/M the p53R2 subunit been identified as a DNA RNR subunit in mammalian cells, the of human cancers with or be to be of this which to the repair of damaged We that cells this by the R2 subunit as a for p53R2 to supply dNTPs for DNA repair. Thus, findings that p53(–/–) HCT-116 cells that not undergo G1 arrest by DNA damage no degradation but a increase in the level of the R2 protein increase in the level of the R2 subunit is correlated with the of G1 arrest and a increase in the of cells in the S and G2/M not the R2 protein is A. Thelander L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The induction of the R2 protein following DNA damage have a the survival of p53(–/–) HCT-116 cells. cytotoxicity is to the of DNA 1985; Google Scholar). Thus, the sensitivity to cisplatin the ability of cells to this DNA through repair A. J. Cancer PubMed Scopus Google Scholar, T. T. G. A. K.H. Y. Cancer Res. 2002; Google Scholar). pronounced increase in the R2 protein in p53(–/–) cells following drug-induced DNA damage that an elevation in the activity of RNR occurs with an enhanced ability to repair damaged is by that DNA damage by cisplatin causes a marked increase in dNTP findings by have shown in and other cell DNA damage by a variety of causes an increase in dNTP levels that in enhanced survival (2Kunz B.A. Kohalmi S.E. Kunkel T.A. Mathews C.K. McIntosh E.M. Reidy J.A. Mutat. Res. 1994; 318: 1-64Crossref PubMed Scopus (78) Google Scholar, A. B. R. Thelander L. Full Text Full Text PDF PubMed Scopus Google Scholar). the of RNA interference to the R2 subunit, results that the level of the R2 protein is correlated with the levels of dNTPs and the sensitivity of cells to cisplatin in p53(–/–) HCT-116 cells. findings the that the level of the R2 protein is a of the ability of the cells to in DNA repair and, is a of the sensitivity to DNA Ectopic expression of p53R2 partially reversed the of p53(–/–) HCT-116 cells to DNA damage caused by that R2-RNR and can be for supplying dNTPs in the of DNA repair. However, the of the RNR inhibitors cells not be by the expression of p53R2. in the ability of p53R2 to the cytotoxicity of be in to two p53R2 not be fully capable of supplying the dNTPs for DNA results that R2 can for p53R2 in DNA repair. it is that p53R2 is to inhibition by and hydroxyurea. R2 and p53R2 in their (9Guittet O. Hakansson P. Voevodskaya N. Fridd S. Graslund A. Arakawa H. Nakamura Y. Thelander L. J. Biol. Chem. 2001; 276: 40647-40651Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and subunits and a that is by inhibitors of RNR M. Graslund A. Thelander L. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar). shown that and R2-RNR are to Triapine, whereas R2-RNR is more to hydroxyurea in J. B. L. W. B. Y. Cancer Res. 64: PubMed Scopus Google Scholar). The important role of RNR in DNA synthesis and repair it a target for and findings that cancer cells with relatively low levels of the R2 protein are more to DNA-damaging and to inhibitors of Thus, of the R2 protein by or the of two of agents. In with this a by et al. B. Liu L. D. W. J. Y. Cancer Res. Google Scholar) shown that the expression of R2 cells to RNA results a positive correlation between the levels of R2 and the sensitivity to DNA damage in cells with These findings the of RNR inhibitors in conjunction with DNA-damaging for cancer The results the of R2-targeted Y. A. N. H. M. H. K. Liu J. A. Cancer Res. Google Scholar). an the of RNR inhibitors the of the of DNA-damaging agents. We for with cell We for an to the