In vitro and In vivo Radiosensitization Inducedby the Ribonucleotide Reductase InhibitorTriapine (3-Aminopyridine- 2-Carboxaldehyde-Thiosemicarbazone) ChristopherA.Barker, 1 William E. Burgan, 2 DonnaJ.Carter, 2 David Cerna, 2 David Gius, 1 Melinda G. Hollingshead, 3 Kevin Camphausen, 1 and PhilipJ.Tofilon 2 Abstract Purpose: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors,Triapine potentlyinhibitsbothRRholoenzymes.Therefore,theeffectsofTriapineontumorcellradiosensi- tivitywereinvestigated. Experimental Design: The effects ofTriapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects ofTriapine on in vivo tumor radiosensitivity.The levels of the RR subunitsweredeterminedusingimmunoblotanalysisandDNAdamageandrepairwereevaluated using gH2AXfoci. Results: ExposureofthetumorcelllinestoTriapinebeforeorimmediatelyafterirradiationresulted in an increase in radiosensitivity. In contrast,Triapine enhanced the radiosensitivity of the normal fibroblastcelllineonlywhentheexposurewasbeforeirradiation.Therewerenoconsistentdiffer- ences between cell lines with respect to the expression of the RR subunits.WhereasTriapine had no effect on radiation-induced gH2AX foci at 1hour, the number of gH2AX foci per cell was significantly greater in theTriapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage.Triapine administration to mice bearing tumor xenografts immediatelyafterirradiationresultedinagreaterthanadditiveincreaseinradiation-inducedtumor growthdelay. Conclusions: These results indicate thatTriapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effectinvolves aninhibitionof DNA repair. Radiotherapy continues to be a primary cancer treatment modality. Recent strategies aimed at increasing its efficacy have focused on targeting the molecules and processes that serve as determinantsofcellularradiosensitivity.Amongthefundamen- talprocessesthatinfluenceradiation-inducedcelldeathisDNA repair; a critical molecule in this process is ribonucleotide reductase(RR).RRcatalyzesthereductionofribonucleotidesto deoxyribonucleotidesandthusprovidesanessentialcomponent for DNA synthesis and repair. RR is composed of two homo- dimersubunits(1).TheR1subunit(composedoftwomolecules of hRRM1) contains the ribonucleotide binding sites and allosteric effector sites. The R2 subunit, which contains a non- hemeironcomplexedwithatyrosylfreeradicalandisessential for catalytic activity, was initially defined as a homodimer of hRRM2. However, Tanaka et al. (2) recently identified the hRRM2 homologue, p53R2, which can serve as a functional alternative to hRRM2 as the R2 subunit (3). The circumstances thatdictatewhethertheR2subunitofRRiscomposedofhRRM2 orp53R2havenotbeencompletelydefined. With respect to radiosensitivity, Kuo et al. (4) showed that whereas the overexpression of the R1 subunit has no effect on radiosensitivity, overexpression of the R2 subunit (in the form of hRRM2) protects against radiation-induced cell death, consistent with the R2 subunit of RR serving as a potential target for radiosensitization. Although a number of agents have been developed as RR inhibitors, hydroxyurea has received the most attention in preclinical and clinical studies. Hydroxyurea quenches the tyrosyl radical thereby destabilizing the iron center of hRRM2 with a resulting loss of RR enzymatic activity (5). A number of groups have reported that hydroxyurea can enhance the radiosensitivity of tumor cell lines (6–9). However, the combination of this RR inhibitor with radiother- apy has achieved only limited success in clinical trials (10). Recently, Shao et al. (11) showed that although hydroxyurea Cancer Therapy: Preclinical Authors’Affiliations: 1 Radiation Oncology Branch, 2 Molecular Radiation Therapeutics Branch, and 3 DevelopmentalTherapeutics Program, National Cancer Institute,Bethesda,Maryland Received1/3/06;revised2/9/06;accepted2/24/06. Grant support: Howard Hughes Medical Institute-NIHResearch Scholars Program(C.A.Barker). Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely toindicate this fact. Requestsforreprints: PhilipJ.Tofilon, Molecular RadiationTherapeutics Branch, Radiation Research Program, EPN/6015A, 6130 Executive Boulevard, MSC 7440, Rockville, MD 20892-7440. Phone: 301-496-6336; Fax: 301-480-5785; E-mail: tofilonp@mail.nih.gov F 2006AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-05-2860 www.aacrjournals.org ClinCancerRes2006;12(9)May1,2006 2912 Research. on April 13, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from