This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shao, J. Articles by Yen, Y. Search for Related Content PubMed PubMed Citation Articles by Shao, J. Articles by Yen, Y.

In Vitro Characterization of Enzymatic Properties and Inhibition of the p53R2 Subunit of Human Ribonucleotide Reductase

¹Departments of Medical Oncology and Therapeutic Research and ²Bioinformatics, City of Hope National Medical Center, Duarte, California, and ³Department of Basic Medical Science, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
p53R2 is a newly identified subunit of ribonucleotide reductase (RR) and plays a crucial role in supplying precursors for DNA repair in a p53-dependent manner. In our current work, all three human RR subunit proteins (p53R2, hRRM2, and hRRM1) were prokaryotically expressed and highly purified. Using an in vitro [³H]CDP reduction assay, the activity of RR reconstituted with either p53R2 or hRRM2 was found to be time, concentration, and hRRM1 dependent. The kinetic activity of p53R2-containing RR was about 20–50% lower than that of hRRM2-containing RR. Using a synthetic heptapeptide to inhibit RR activity, it was shown that p53R2 bound to hRRM1 through the same COOH-terminal heptapeptide as hRRM2. However, hRRM2 had a 4.76-fold higher binding affinity for hRRM1 than p53R2, which may explain the reduced RR activity of p53R2 relative to hRRM2. Of interest, p53R2 was 158-fold more susceptible to the iron chelator deferoxamine mesylate than hRRM2, although the iron content of the two proteins determined by atomic absorption spectrometer was almost the same. To the contrary, p53R2 was 2.50-fold less sensitive than hRRM2 to the radical scavenger hydroxyurea, whereas EPR showed similar spectra of the tyrosyl radical in two proteins. Triapine, a new RR inhibitor, was equally potent for p53R2 and hRRM2. These inhibition studies showed that the iron center and tyrosyl radical are involved in RR activity for both p53R2 and hRRM2. The susceptibility differences to RR inhibitors between p53R2 and hRRM2 may lead to a new direction in drug design for human cancer treatment.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Ribonucleotide reductase (RR) is the unique enzyme responsible for the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides (dNTPs), which are the building blocks for DNA replication and repair in all living cells (1) . Human RR is a tetramer complex, composed of two non-identical homodimers, hRRM1 and hRRM2 (R1and R2 in mouse)_. The large subunit hRRM1 (87 kD) harbors the catalytic site, allosteric effector-binding sites, and redox active disulfides that participate in the reduction of substrates, whereas the small subunit hRRM2 (43 kD) contains an oxygen-linked di-ferric center and one tyrosyl radical per monomer that are essential for enzymatic activity (1 , 2) .

p53R2, a 351-amino acid peptide (39 kD), is a newly identified hRRM2 homolog (80%) and plays a crucial role in supplying dNTPs for DNA repair (3 , 4) . Several known functional domains in mouse R2 are also conserved in p53R2 (2, 3, 4, 5, 6, 7) , including the iron ligands, the radical site tyrosine, the hydrophobic pocket surrounding the tyrosyl radical site, the radical transfer pathway from small subunit to large subunit, the COOH-terminal sequence for binding to large subunit, and the hydrophobic channel from the surface to the interior of the protein. However, the actual functions of these domains in p53R2 for RR activity are not confirmed. The major sequence difference between the two small subunits is that p53R2 lacks 33 amino acid residues in its NH₂ terminus (8) . The human p53R2 gene contains a p53-binding site in intron 1. The expression of p53R2, but not hRRM2, is induced by UV light, -irradiation, or DNA-damaging agents in a p53-dependent manner (3 , 4) . p53R2 forms an active RR in vitro with R1, and the expression of the R1 is induced in resting cells after UV irradiation (9) . These findings lead to the hypothesis that there are two pathways in human cells to supply dNTPs for DNA synthesis: one through the activity of hRRM2, involved in normal maintenance of dNTPs for DNA replication during the S-phase in a cell cycle-dependent manner, and the other through p53R2, supplying dNTPs for DNA repair in G₀-G₁ cells in a p53-dependent manner (10 , 11) . Recently, our lab found that in p53 mutant and null cells, hRRM2 complements p53R2 in response to UV-induced DNA repair (12) . Furthermore, p53 can interact with p53R2 and hRRM2 at the protein level to regulate RR activity (13) . However, it is still unclear how the expression and activity of the two small subunits are coordinately regulated in cells. It has been shown that alterations in RR levels, in particular overexpression of hRRM2, can have significant effects on the biological properties of cells, tumor development and metastasis, and drug resistance (14 , 15) . A study of Rrm2b (encoding p53R2)-null mice showed that impairment of the p53R2-involved DNA repair pathway enhances the frequency of spontaneous mutations and activates p53-dependent apoptotic pathway(s) (16) . However, the role that p53R2 plays in the development of human cancers remains to be elucidated.

Because RR is overexpressed in cancer cells, the development of RR inhibitors may prove useful as anticancer drugs. A number of compounds have been characterized as RR inhibitors. The first category targets hRRM2 by quenching the tyrosyl radical and/or affecting the iron center, such as hydroxyurea (17) , 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (triapine), and deferoxamine mesylate (DFO; Ref. 18 ). The second category of drugs targets hRRM1 by disturbing binding of allosteric effectors or using nucleoside analogues, such as 2,2'-difluorodeoxycytidine (19) . The third category includes peptidomimetic drugs that competitively inhibit the binding of the small subunit to the large subunit (20) and inhibitors that disturb the radical transfer pathway (7) . p53R2, functioning in a p53-dependent DNA repair pathway, may have the potential to be considered as a new therapeutic target for human cancer (7 , 11) . Furthermore, because p53R2 and hRRM2 play different roles in cells, inhibitors specific for each subunit could have different clinical values. However, no p53R2 inhibitors have been characterized thus far.

In our current work, a simple and rapid procedure was developed for the expression and purification of all three subunits of RR. Using an in vitro RR assay, we examined the enzymatic properties and mechanisms of p53R2 including its interaction with the large subunit and its response to RR inhibitors. These findings may provide a basis for the design and screening of more effective and subunit-selective RR inhibitors for human cancer therapy.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Plasmid Construction.
Total RNA was isolated from human oropharyngeal carcinoma KB cells. The coding sequences of human p53R2, hRRM2, and hRRM1 proteins were obtained by reverse transcription-PCR using the following primer pairs: (1) hRRM1: 5'-CAGCGGCCGCGATGCATGTGATCAAGCGA (with a NotI enzyme digestion site) and 3'-AAGCGGCCGCTCAGGATCCACACATCAG (with a NotI enzyme digestion site); (2) hRRM2: 5'-ATCCGGATCCACTATGCTCTCCCTCCGTGT (with a BamHI enzyme digestion site) and 3'-GCTTAAGCTTATTTAGAAGTCAGCATCCAAG (the NotI enzyme digestion site comes from pDrive vector); and (3) p53R2: 5'-TCGGATCCATGGGCGACCCGGAAAGG (with a BamHI enzyme digestion site) and 3'-GCGGCCGCTTAAAAATCTGCATCCAA (with a NotI enzyme digestion site). The PCR products were purified and inserted into the pDrive cloning vector (Qiagen, Valencia, CA) via U-A ligation. After restriction digestion, the coding sequence of each RR subunit was then cloned in-frame with an NH₂-terminal 6x His-tag into the prokaryotic expression vector pET28 (Novagen, Madison, WI). All constructs (pET28a-p53R2, pET28a-hRRM2, and pET28b-hRRM1) were verified by DNA sequencing.

Protein Expression and Purification.
Three recombinant RR subunit proteins were expressed from BL21 (DE3) bacteria (Stratagene, La Jolla, CA) and were purified using Ni-resin (Qiagen) affinity chromatography based on Qiagen’s protocol. For p53R2 protein, an overnight culture of the transformed bacteria was diluted 50-fold in 1 liter of Luria broth medium containing 30 µg/ml kanamycin and grown at 37°C for 4 h and then induced by 1 mM isopropyl-1-thio-ß-D-galactopyranoside for an additional 3 h at 30°C. After harvesting by centrifugation, the cell pellets were disrupted by incubation with Bugbuster and benzonase nuclease (Qiagen) at 4°C for 30 min with vigorous agitation, and the lysate was centrifuged at 16,000 x g for 30 min at 4°C. The clear supernatant was incubated with Ni-resin at 4°C for 30 min, loaded on a column, washed with at least 30-fold bed volume of 50 mM NaH₂PO₄, 800 mM NaCl, 50 mM imidazole, pH 7.0, 0.1% Triton X-100, and 10 mM 2-mercaptoethanol, and finally eluted with 50 mM NaH₂PO₄, 300 mM NaCl, and 125 mM imidazole, pH 7.0. The eluates were thoroughly dialyzed against 2000 sample volumes of 25 mM Tris-HCl, pH 7.4, at 4°C overnight. Expression and purification of the hRRM2 and hRRM1 proteins were performed in the same way as for p53R2. The proteins were placed in aliquots and frozen at -70°C. The RR activity of the two small subunit proteins was stable within a 5-month period, and after that, the activity of p53R2 gradually decreased.

Qualitative and Quantitative Analysis of Protein Preparations.
Protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). Protein purity was determined by densitometric scanning of the Coomassie-stained SDS-PAGE gel. For immunoblotting, protein samples were analyzed using goat anti-p53R2, hRRM2, and hRRM1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). For MS/MS (tandem mass spectrometry), protein bands were cut from a 10% SDS-PAGE gel, in-gel digested with trypsin, and examined by HPLC/MS/MS and SEQUEST searching at the City of Hope National Medical Center Mass Spectrometry Core Facility.

Atomic Absorption Spectrometry.
A Perkin-Elmer atomic absorption spectrometer equipped with a HGA850 graphite furnace was used for measurement of iron content at the City of Hope National Medical Center Pharmacoanalytic Core Facility. PE Pure Iron standard solution (Perkin-Elmer, Norwalk, CT) was 2-fold serially diluted from 50–1000 µg/l to construct the standard curve (y = 0.00052 x + 0.0027; x, concentration; y, absorbance; r = 0.993). The purified proteins were thoroughly dialyzed and diluted 1:5 with demineralized water. Iron content was expressed as µg of iron per µmol of each RR subunit protein.

EPR (Electron Paramagnetic Resonance) Spectra.
X-band EPR spectra were measured with a Bruker EMX spectrometer equipped with an Oxford helium cryostat at the Department of Chemistry of The California Institute of Technology. Purified samples of p53R2 and hRRM2 proteins were frozen in liquid nitrogen before insertion in the cavity. Instrumental parameters were: T, 20 K; microwave frequency, 9.376 GHz; microwave power, 0.5 mW; modulation amplitude, 4 gauss; and modulation frequency, 100 KHz.

RR Activity Assay in Vitro.
RR activity was measured using the [³H]CDP reduction assay as described previously with a slight modification (17) . Briefly, the reaction mixture contained 0.125 µM [³H]CDP, 50 mM HEPES (pH 7.2), 6 mM DTT, 4 mM magnesium acetate, 2 mM ATP, 0.05 mM CDP, 100 mM KCl, 0.24 mM NADPH, and different amounts of purified proteins in a final volume of 100 µl. After incubation at 37°C for a desired time and dephosphorylation, samples were analyzed by HPLC and liquid scintillation counting. To determine enzymatic specific activity, p53R2 or hRRM2 was 2-fold serially diluted (0.625–10 µg) and incubated with 30 µg of hRRM1 at 37°C for 15 min. Specific activity was calculated as nmol of dCDP formed/min/mg protein.

RR Inhibitor Preparation and Test.
The heptapeptide Ac-FTLDADF was synthesized by the City of Hope National Medical Center DNA/RNA/Peptide Synthesis Facility. DFO and hydroxyurea were obtained from Sigma. Triapine was a gift from Vion Pharmaceutical (New Haven, CT). For assays of the inhibition of RR activity, each inhibitor was serially diluted in 25 mM Tris-HCl (pH 7.4), incubated with purified RR subunit proteins at room temperature for 30 min, added directly to the reaction buffer, and analyzed for RR activity as described above. RR activity in the absence of any inhibitor was used as a control. Results are expressed as a percentage of the control value (relative RR activity). IC₅₀s were determined by interpolation of the plotted data to show the inhibitor concentration that produced 50% inhibition of RR activity.

Statistics.
Results are presented as the mean ± SD of three separate experiments, each performed in duplicate. The significance of the data was determined using Student’s t test (two-tail). P < 0.01 was deemed significant.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Expression and Purification of Three Human RR Subunit Proteins.
Human p53R2, hRRM2, and hRRM1 proteins were all expressed using the pET28-BL21(DE3) prokaryotic system except that different restriction sites were used for each construction and were purified to 90% purity using one-step Ni-resin affinity chromatography (Fig. 1) . Protein yields were 20, 10, and 2 mg/l of induced bacterial culture for p53R2, hRRM2, and hRRM1 proteins. Reconstitution of RR activity was straightforward by mixing purified p53R2 or hRRM2 with hRRM1 and did not require regeneration of the iron-tyrosyl radical center that is necessary for RR activity (9 , 21) . Use of the same expression and purification system for all three subunits greatly increased the comparability of enzyme activity between the two small subunits. A slightly smaller band copurified with the 42.5 kD His-tagged p53R2 band regardless of the purification stringency or the use of protease inhibitors. Both bands were examined by HPLC/MS/MS. SEQUEST matching confirmed that both bands belonged to human p53R2 with 65 and 48% sequence coverage for the major and the smaller bands, respectively. Therefore, the small band may represent partially degraded p53R2 preparation.

View larger version (46K): [in this window] [in a new window]   Fig. 1. In vitro expression and purification of human RR subunits. A, 10% SDS-PAGE analysis of the purified recombinant proteins by one-step Ni-resin affinity chromatography. Lane 1, molecular mass marker; Lane 2, hRRM1 (5 µg); Lane 3, hRRM2 (2.5 µg); Lane 4, p53R2 (2.5 µg). B, immunoblot confirmation of protein preparations.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Activity of RR holoenzyme reconstituted with human p53R2 or hRRM2. 2.5 µg (A) or 5 µg (B) of p53R2 or hRRM2 was mixed with 20 µg of hRRM1 and assayed for RR activity. Samples were incubated at 37°C for the indicated times, and RR activity was assayed as described in "Materials and Methods." C, hRRM1 was 2-fold serially diluted (0.938–15 µg) and incubated with an excess of p53R2 or hRRM2 (25 µg) at 37°C for 30 min and then assayed for RR activity. The data are presented as the means of three separate experiments, each performed in duplicate; bars, SD. *, statistical significance (P < 0.01).

View larger version (44K): [in this window] [in a new window]   Fig. 3. Binding affinity of human p53R2 and hRRM2 to hRRM1. A, homology model of human p53R2 monomer (288 residues from E26 to E313) created based on crystal structure of mouse R2 template (PDB code: 1H0N, by Strand, K. R. et al.). Tyr-138 is red. The two iron ions are shown as blue spheres. The iron ligands are yellow. B, crystal structure of R1 monomer with the COOH-terminal peptide (20 residues) of R2 from Escherichia coli (PDB code: 4R1R, by Eriksson, M. et al.). The COOH-terminal peptide is highlighted. This figure shows how the COOH terminus of the small subunit interacts with the large subunit. The catalytic site and the allosteric effector-binding sites (specificity and activity site) in the large subunit also are outlined. Figures in A and B were generated using SYBYL (Tripos) module Composer. C, competitive inhibition of RR activity by the synthetic heptapeptide. The synthetic heptapeptide was 2-fold serially diluted (3.91–500 µM) and incubated with 5 µg of hRRM2 or p53R2 (1.2 µM) in the presence of 5 µg of hRRM1 (0.6 µM) and then assayed for RR activity as described in "Materials and Methods." The data are presented as the means of three separate experiments, each performed in duplicate; bars, SD. *, statistical significance (P < 0.01).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Response of human p53R2 and hRRM2 to RR inhibitors. A, inhibition curve of DFO (5-fold serially diluted from 2.40 µM to 37.5 mM). B, EPR spectra of the tyrosyl radical in human p53R2 (a) and hRRM2 (b) proteins. The spectra were recorded as described in "Materials and Methods." C, inhibition curve of hydroxyurea (2-fold serially diluted from 78.1 µM to 5 mM). D, inhibition curve of triapine (5-fold serially diluted from 16 nM to 10 µM). Each serially diluted inhibitor was incubated with 2.5 µg of hRRM2 or p53R2 and 10 µg of hRRM1 and assayed for RR activity as described in "Materials and Methods." The data are presented as the means of three separate experiments, each performed in duplicate; bars, SD. *, statistical significance (P < 0.01).

Triapine is a new RR inhibitor currently being tested in phase II clinical trials for cancer therapy (24) . Our results showed that triapine directly inactivated the RR activity of p53R2 and hRRM2 with almost equal efficiency (Fig. 4D) . The IC₅₀ of triapine was 112 ± 8.83 nM for p53R2 and 144 ± 7.63 nM for hRRM2. Of the three RR inhibitors tested in this study, triapine was the most potent (Table 1) . This result is consistent with the data comparing the efficiency of RR inhibitors in cancer cells (18 , 24 , 25) . The antiproliferative activity of triapine in intact cells involves multiple inhibitory mechanisms. It not only has iron chelation efficacy that is similar to that of DFO but is capable of generating reactive oxygen species after binding iron that may result in destruction of the tyrosyl radical (24 , 25) . It is known that the low efficiency of hydroxyurea is associated with its physicochemical properties, e.g., very high hydrophilicity and small molecular size (26) . In contrast, the hydrophobicity and structural feature of triapine may also contribute to its marked effect on RR activity.

	ACKNOWLEDGMENTS

 
The EPR measurements were performed at the Department of Chemistry of The California Institute of Technology. We thank Dr. Angel J. Di Bilio for discussions. We thank L. Su and K. Karlsberg for comments and graphical support for the manuscript.

	FOOTNOTES

 
Grant support: NCI R01 Grant CA72767; partially supported by Sino America Cancer Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Yun Yen, Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. Phone: (626) 359-8111, extension 62867; Fax: (626) 301-8233; E-mail: yyen{at}coh.org

Received 9/26/03. Revised 10/31/03. Accepted 11/ 3/03.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Jordan A., Reichard P. Ribonucleotide reductases. Annu. Rev. Biochem., 67: 71-98, 1998.[CrossRef][Medline]
2. Kauppi B., Nielsen B. B., Ramaswamy S., Larsen I. K., Thelander M., Thelander L., Eklund H. The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2. J. Mol. Biol., 262: 706-720, 1996.[CrossRef][Medline]
3. Tanaka H., Arakawa H., Yamaguchi T., Shiraishi K., Fukuda S., Matsui K., Takei Y., Nakamura Y. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature (Lond.), 404: 42-49, 2000.[CrossRef][Medline]
4. Nakano K., Balint E., Ashcroft M., Vousden K. H. A ribonucleotide reductase gene is a transcriptional target of p53 and p73. Oncogene, 19: 4283-4289, 2000.[CrossRef][Medline]
5. Rova U., Adrait A., Potsch S., Graslund A., Thelander L. Evidence by mutagenesis that Tyr³⁷⁰ of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway. J. Biol. Chem., 274: 23746-23751, 1999.[Abstract/Free Full Text]
6. Adrait A., Ohrstrom M., Barra A. L., Thelander L., Graslund A. EPR studies on a stable sulfinyl radical observed in the iron-oxygen-reconstituted Y177F/I263C protein R2 double mutant of ribonucleotide reductase from mouse. Biochemistry, 41: 6510-6516, 2002.[CrossRef][Medline]
7. Eklund H., Uhlin U., Farnegardh M., Logan D. T., Nordlund P. Structure and function of the radical enzyme ribonucleotide reductase. Prog. Biophys. Mol. Biol., 77: 177-268, 2001.[CrossRef][Medline]
8. Chabes A., Pfleger C. M., Kirschner M. W., Thelander L. Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis. Proc. Natl. Acad. Sci. USA, 100: 3925-3929, 2003.[Abstract/Free Full Text]
9. Guittet O., Hakansson P., Voevodskaya N., Fridd S., Graslund A., Arakawa H., Nakamura Y., Thelander L. Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells. J. Biol. Chem., 276: 40647-40651, 2001.[Abstract/Free Full Text]
10. Yamaguchi T., Matsuda K., Sagiya Y., Iwadate M., Fujino M. A., Nakamura Y., Arakawa H. p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint. Cancer Res., 61: 8256-8262, 2001.[Abstract/Free Full Text]
11. Lozano G., Elledge S. J. p53 sends nucleotides to repair DNA. Nature (Lond.), 404: 24-25, 2000.[CrossRef][Medline]
12. Zhou B., Liu X., Mo X., Xue L., Darwish D., Qiu W., Shih J., Hwu E. B., Luh F., Yen Y. The human ribonucleotide reductase subunit hRRM2 complements p53R2 in response to UV-induced DNA repair in cells with mutant p53. Cancer Res., 63: 6583-6594, 2003.[Abstract/Free Full Text]
13. Xue L., Zhou B., Liu X., Qiu W., Jin Z., Yen Y. Wild-type p53 regulates human ribonucleotide reductase by protein-protein interaction with p53R2 as well as hRRM2 subunits. Cancer Res., 63: 980-986, 2003.[Abstract/Free Full Text]
14. Fan H., Villegas C., Huang A., Wright J. A. The mammalian ribonucleotide reductase R2 component cooperates with a variety of oncogenes in mechanisms of cellular transformation. Cancer Res., 58: 1650-1653, 1998.[Abstract/Free Full Text]
15. Zhou B. S., Tsai P., Ker R., Tsai J., Ho R., Yu J., Shih J., Yen Y. Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential. Clin. Exp. Metastasis, 16: 43-49, 1998.[CrossRef][Medline]
16. Kimura T., Takeda S., Sagiya Y., Gotoh M., Nakamura Y., Arakawa H. Impaired function of p53R2 in Rrm2b-null mice causes severe renal failure through attenuation of dNTP pools. Nat. Genet., 34: 440-4445, 2003.[CrossRef][Medline]
17. Yen Y., Grill S. P., Dutschman G. E., Chang C. N., Zhou B. S., Cheng Y. C. Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine. Cancer Res., 54: 3686-3691, 1994.[Abstract/Free Full Text]
18. Richardson D. R. Iron chelators as therapeutic agents for the treatment of cancer. Crit. Rev. Oncol. Hematol., 42: 267-281, 2002.[Medline]
19. Goan Y. G., Zhou B., Hu E., Mi S., Yen Y. Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. Cancer Res., 59: 4204-4207, 1999.[Abstract/Free Full Text]
20. Cooperman B. S. Oligopeptide inhibition of class I ribonucleotide reductases. Biopolymers, 71: 117-131, 2003.[Medline]
21. Mann G. J., Graslund A., Ochiai E., Ingemarson R., Thelander L. Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry, 30: 1939-1947, 1991.[CrossRef][Medline]
22. Nyholm S., Mann G. J., Johansson A. G., Bergeron R. J., Graslund A., Thelander L. Role of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron chelators. J. Biol. Chem., 268: 26200-26205, 1993.[Abstract/Free Full Text]
23. Zhou B. S., Hsu N. Y., Pan B. C., Doroshow J. H., Yen Y. Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells. Cancer Res., 55: 1328-1333, 1995.[Abstract/Free Full Text]
24. Finch R. A., Liu M. C., Cory A. H., Cory J. G., Sartorelli A. C. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): an inhibitor of ribonucleotide reductase with antineoplastic activity. Adv. Enzyme Regul., 39: 3-12, 1999.[CrossRef][Medline]
25. Chaston T. B., Lovejoy D. B., Watts R. N., Richardson D. R. Examination of the antiproliferative activity of iron chelators: multiple cellular targets and the different mechanism of action of triapine compared with desferrioxamine and the potent pyridoxal isonicotinoyl hydrazone analogue 311. Clin. Cancer Res., 9: 402-414, 2003.[Abstract/Free Full Text]
26. Ren S., Wang R., Komatsu K., Bonaz-Krause P., Zyrianov Y., McKenna C. E., Csipke C., Tokes Z. A., Lien E. J. Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents. J. Med. Chem., 45: 410-419, 2002.[Medline]

This article has been cited by other articles:

				J. Wang, G. J. S. Lohman, and J. Stubbe Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases PNAS, September 4, 2007; 104(36): 14324 - 14329. [Abstract] [Full Text] [PDF]

				S. Y. Rha, H. C. Jeung, Y. H. Choi, W. I. Yang, J. H. Yoo, B. S. Kim, J. K. Roh, and H. C. Chung An Association Between RRM1 Haplotype and Gemcitabine-Induced Neutropenia in Breast Cancer Patients Oncologist, June 1, 2007; 12(6): 622 - 630. [Abstract] [Full Text] [PDF]

				Y. Yu, J. Wong, D. B. Lovejoy, D. S. Kalinowski, and D. R. Richardson Chelators at the Cancer Coalface: Desferrioxamine to Triapine and Beyond Clin. Cancer Res., December 1, 2006; 12(23): 6876 - 6883. [Abstract] [Full Text] [PDF]

				X. Liu, B. Zhou, L. Xue, J. Shih, K. Tye, W. Lin, C. Qi, P. Chu, F. Un, W. Wen, et al. Metastasis-Suppressing Potential of Ribonucleotide Reductase Small Subunit p53R2 in Human Cancer Cells. Clin. Cancer Res., November 1, 2006; 12(21): 6337 - 6344. [Abstract] [Full Text] [PDF]

				C. A. Barker, W. E. Burgan, D. J. Carter, D. Cerna, D. Gius, M. G. Hollingshead, K. Camphausen, and P. J. Tofilon In vitro and In vivo Radiosensitization Induced by the Ribonucleotide Reductase Inhibitor Triapine (3-Aminopyridine-2-Carboxaldehyde-Thiosemicarbazone). Clin. Cancer Res., May 1, 2006; 12(9): 2912 - 2918. [Abstract] [Full Text] [PDF]

				J. Shao, B. Zhou, A. J. Di Bilio, L. Zhu, T. Wang, C. Qi, J. Shih, and Y. Yen A Ferrous-triapine complex mediates formation of reactive oxygen species that inactivate human ribonucleotide reductase. Mol. Cancer Ther., March 1, 2006; 5(3): 586 - 592. [Abstract] [Full Text] [PDF]

				L. Xue, B. Zhou, X. Liu, T. Wang, J. Shih, C. Qi, Y. Heung, and Y. Yen Structurally Dependent Redox Property of Ribonucleotide Reductase Subunit p53R2 Cancer Res., February 15, 2006; 66(4): 1900 - 1905. [Abstract] [Full Text] [PDF]

				D. S. Kalinowski and D. R. Richardson The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer Pharmacol. Rev., December 1, 2005; 57(4): 547 - 583. [Abstract] [Full Text] [PDF]

				B. Zhou, J. Shao, L. Su, Y.-C. Yuan, C. Qi, J. Shih, B. Xi, B. Chu, and Y. Yen A dityrosyl-diiron radical cofactor center is essential for human ribonucleotide reductases Mol. Cancer Ther., December 1, 2005; 4(12): 1830 - 1836. [Abstract] [Full Text] [PDF]

				Z. P. Lin, M. F. Belcourt, J. G. Cory, and A. C. Sartorelli 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 J. Biol. Chem., June 25, 2004; 279(26): 27030 - 27038. [Abstract] [Full Text] [PDF]

				S. Yamaoka, M. Miyaji, T. Kitano, H. Umehara, and T. Okazaki Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells J. Biol. Chem., April 30, 2004; 279(18): 18688 - 18693. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shao, J. Articles by Yen, Y. Search for Related Content PubMed PubMed Citation Articles by Shao, J. Articles by Yen, Y.