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Objective: Ribonucleotide reductase (RNR) supplies deoxyribonucleotide diphosphates demanded by cells to repair radiation-induced DNA damage. Here, we investigate the impact of pretherapy RNR M1, M2, and M2b (p53R3) subunit level upon human cervical cancer radiochemosensitivity., Materials/methods: Immunohistochemistry was performed on a tissue array comprised of 18 paired benign uterine cervix and stage IB2 cervical cancers to evaluate the relationship between cytosolic RNR M1, M2, and M2b staining intensity and radiochemotherapy cancer response. Patients underwent surgical hysterectomy (n = 8), or daily radiation (45 Gy), coadministered once-weekly cisplatin (40 mg/m), then low-dose rate brachytherapy (30 Gy) followed by adjuvant hysterectomy (n = 10). Radiochemotherapy response was determined by Response Evaluation Criteria In Solid Tumors version 1.0 criteria during brachytherapy. Cancer relapse rates and disease-free survival were calculated., Results: M1, M2, and M2b antibody staining intensity was low (0-1+) in benign uterine cervical tissue. M1 and M2b immunoreactivity was 2+ or 3+ in most (13/18) cervical cancers. M2 immunoreactivity was 3+ in nearly all (16/18) cervical cancers. Cervical cancers overexpressing M1 and M2b had an increased hazard for incomplete radiochemotherapy response, relapse, and shortened disease-free survival., Conclusions: Ribonucleotide reductase subunit levels may predict human cervical cancer radiochemosensitivity and subsequent posttherapy cancer outcome. Further validation testing of RNR subunits as biomarkers for radiochemotherapy response is warranted.

Background: The potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer., Methods: Preclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP (96 mg/m(2), day one to four) and cisplatin (25 mg/m(2), day two and three) infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response., Results: 3-AP preceding cisplatin exposure in platinum-resistant ovarian cancer cells was not as effective as sequencing cisplatin plus 3-AP together in cell survival assays. Platinum-mediated DNA damage (i.e., γH2AX foci) resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual., Conclusions: When sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/m(2)) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron., Trial Registry: ClinicalTrials.gov NCT00081276.

The anticancer agent 3-aminopyridine-2-carboxaldehyde thiosemicarbazone is a ribonucleotide reductase inhibitor. It inactivates ribonucleotide reductase by disrupting an iron-stabilized radical in ribonucleotide reductase's small subunits, M2 and M2b (p53R2). Unfortunately, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone also alters iron II (Fe(2+)) in hemoglobin. This creates Fe(3+) methemoglobin that does not deliver oxygen. Fe(2+) in hemoglobin normally auto-oxidizes to inactive Fe(3+) methemoglobin at a rate of nearly 3% per day and this is counterbalanced by a reductase system that normally limits methemoglobin concentrations to less than 1% of hemoglobin. This balance may be perturbed by symptomatic toxicity levels during 3-aminopyridine-2-carboxaldehyde thiosemicarbazone therapy. Indications of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone sequelae attributable to methemoglobinemia include resting dyspnea, headaches and altered cognition. Management of methemoglobinemia includes supplemental oxygen, ascorbate and, most importantly, intravenously administered methylene blue as a therapeutic antidote.

Mammalian ribonucleotide reductase (RNR) activity has been reported to be nonmonotonic in ATP. If many nonlinear models are to be fitted to such data automatically as part of a model search process, use of the same initial parameter values across all models can lead to too many poor fitting, monotonic least squares fits, i.e., false model rejections. We propose that such fits can be rescued by using as initial parameter estimates the final estimates of neighboring models that do have nonmonotonic fits; here models are neighbors if complexes that they represent differ by at most one ligand. We use this approach to show that troughs in RNR activity versus ATP can be fitted similarly well by models that do or do not demand a third ATP binding site.

Cells generate 2'-deoxyribonucleoside triphosphates (dNTPs) for both replication and repair of damaged DNA predominantly through de novo reduction of intracellular ribonucleotides by ribonucleotide reductase (RNR). Cells can also salvage deoxynucleosides by deoxycytidine kinase/thymidine kinase 1 in the cytosol or by deoxyguanosine kinase/thymidine kinase 2 in mitochondria. In this study we investigated whether the salvage dNTP supply pathway facilitates DNA damage repair, promoting cell survival, when pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC no. 663249) impairs the de novo pathway. Human cervical cancer cells were subjected to radiation with or without 3-AP under medium deoxynucleoside concentrations of 0, 0.05, 0.5 and 5.0 µM. Efficacy of DNA damage repair was assessed by γ-H2AX flow cytometry and focus counts, by single cell electrophoresis (Comet assay), and by caspase 3 cleavage assay as a marker of treatment-induced apoptosis. Cell survival was assessed by colony formation. We found that deoxyribonucleotide salvage facilitates DNA repair during RNR inhibition by 3-AP and that salvage reduces the radiochemosensitivity of human cervical cancer cells.

Purpose: To test whether pharmacologic inhibition of ribonucleotide reductase (RNR) by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhances radiation sensitivity during low-dose-rate ionizing radiation provided by a novel purpose-built iridium-192 cell irradiator., Methods and Materials: The cells were exposed to low-dose-rate radiation (11, 23, 37, 67 cGy/h) using a custom-fabricated cell irradiator or to high-dose-rate radiation (330 cGy/min) using a conventional cell irradiator. The radiation sensitivity of human cervical (CaSki, C33-a) cancer cells with or without RNR inhibition by 3-AP was evaluated using a clonogenic survival and an RNR activity assay. Alteration in the cell cycle distribution was monitored using flow cytometry., Results: Increasing radiation sensitivity of both CaSki and C33-a cells was observed with the incremental increase in radiation dose rates. 3-AP treatment led to enhanced radiation sensitivity in both cell lines, eliminating differences in cell cytotoxicity from the radiation dose rate. RNR blockade by 3-AP during low-dose-rate irradiation was associated with low RNR activity and extended G(1)-phase cell cycle arrest., Conclusions: We conclude that RNR inhibition by 3-AP impedes DNA damage repair mechanisms that rely on deoxyribonucleotide production and thereby increases radiation sensitivity of human cervical cancers to low-dose-rate radiation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

R is an object oriented free and open source statistical computing environment. The R package Combinatorially Complex Equilibrium Model Selection is being developed to meet the analysis challenges of ribonucleotide reductase (RNR). An example of its use is given here. This example involves ATP-induced R1 hexamerization dynamic light scattering data that suggests that R1 hexamers have two types of a-sites, one that binds ATP and another that does not (here, R1 is the large subunit of RNR).

Background: Ribonucleotide reductase is the main control point of dNTP production. It has two subunits, R1, and R2 or p53R2. R1 has 5 possible catalytic site states (empty or filled with 1 of 4 NDPs), 5 possible s-site states (empty or filled with ATP, dATP, dTTP or dGTP), 3 possible a-site states (empty or filled with ATP or dATP), perhaps two possible h-site states (empty or filled with ATP), and all of this is folded into an R1 monomer-dimer-tetramer-hexamer equilibrium where R1 j-mers can be bound by variable numbers of R2 or p53R2 dimers. Trillions of RNR complexes are possible as a result. The problem is to determine which are needed in models to explain available data. This problem is intractable for 10 reactants, but it can be solved for 2 and is here for R1 and ATP., Results: Thousands of ATP-induced R1 hexamerization models with up to three (s, a and h) ATP binding sites per R1 subunit were automatically generated via hypotheses that complete dissociation constants are infinite and/or that binary dissociation constants are equal. To limit the model space size, it was assumed that s-sites are always filled in oligomers and never filled in monomers, and to interpret model terms it was assumed that a-sites fill before h-sites. The models were fitted to published dynamic light scattering data. As the lowest Akaike Information Criterion (AIC) of the 3-parameter models was greater than the lowest of the 2-parameter models, only models with up to 3 parameters were fitted. Models with sums of squared errors less than twice the minimum were then partitioned into two groups: those that contained no occupied h-site terms (508 models) and those that contained at least one (1580 models). Normalized AIC densities of these two groups of models differed significantly in favor of models that did not include an h-site term (Kolmogorov-Smirnov p < 1 x 10(-15)); consistent with this, 28 of the top 30 models (ranked by AICs) did not include an h-site term and 28/30 > 508/2088 with p < 2 x 10(-15). Finally, 99 of the 2088 models did not have any terms with ATP/R1 ratios >1.5, but of the top 30, there were 14 such models (14/30 > 99/2088 with p < 3 x 10(-16)), i.e. the existence of R1 hexamers with >3 a-sites occupied by ATP is also not supported by this dataset., Conclusion: The analysis presented suggests that three a-sites may not be occupied by ATP in R1 hexamers under the conditions of the data analyzed. If a-sites fill before h-sites, this implies that the dataset analyzed can be explained without the existence of an h-site.

Background: Biochemical equilibria are usually modeled iteratively: given one or a few fitted models, if there is a lack of fit or over fitting, a new model with additional or fewer parameters is then fitted, and the process is repeated. The problem with this approach is that different analysts can propose and select different models and thus extract different binding parameter estimates from the same data. An alternative is to first generate a comprehensive standardized list of plausible models, and to then fit them exhaustively, or semi-exhaustively., Results: A framework is presented in which equilibriums are modeled as pairs (g, h) where g = 0 maps total reactant concentrations (system inputs) into free reactant concentrations (system states) which h then maps into expected values of measurements (system outputs). By letting dissociation constants Kd be either freely estimated, infinity, zero, or equal to other Kd, and by letting undamaged protein fractions be either freely estimated or 1, many g models are formed. A standard space of g models for ligand-induced protein dimerization equilibria is given. Coupled to an h model, the resulting (g, h) were fitted to dTTP induced R1 dimerization data (R1 is the large subunit of ribonucleotide reductase). Models with the fewest parameters were fitted first. Thereafter, upon fitting a batch, the next batch of models (with one more parameter) was fitted only if the current batch yielded a model that was better (based on the Akaike Information Criterion) than the best model in the previous batch (with one less parameter). Within batches models were fitted in parallel. This semi-exhaustive approach yielded the same best models as an exhaustive model space fit, but in approximately one-fifth the time., Conclusion: Comprehensive model space based biochemical equilibrium model selection methods are realizable. Their significance to systems biology as mappings of data into mathematical models warrants their development.

Background: Recent data suggest that ribonucleotide reductase (RNR) exists not only as a heterodimer R12R22 of R12 and R22 homodimers, but also as tetramers R14R24 and hexamers R16R26. Recent data also suggest that ATP binds the R1 subunit at a previously undescribed hexamerization site, in addition to its binding to previously described dimerization and tetramerization sites. Thus, the current view is that R1 has four NDP substrate binding possibilities, four dimerization site binding possibilities (dATP, ATP, dGTP, or dTTP), two tetramerization site binding possibilities (dATP or ATP), and one hexamerization site binding possibility (ATP), in addition to possibilities of unbound site states. This large number of internal R1 states implies an even larger number of quaternary states. A mathematical model of RNR activity which explicitly represents the states of R1 currently exists, but it is complicated in several ways: (1) it includes up to six-fold nested sums; (2) it uses different mathematical structures under different substrate-modulator conditions; and (3) it requires root solutions of high order polynomials to determine R1 proportions in mono-, di-, tetra- and hexamer states and thus RNR activity as a function of modulator and total R1 concentrations., Results: We present four (one for each NDP) rational polynomial models of RNR activity as a function of substrate and reaction rate modifier concentrations. The new models avoid the complications of the earlier model without compromising curve fits to recent data., Conclusion: Compared to the earlier model of recent data, the new rational polynomial models are simpler, adequately fitting, and likely better suited for biochemical network simulations.