Abstract 445: shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation.
Authors: Flanagan Sheryl A. , Cooper Kristin , Mannava Sudha , Nikiforov Mikhail , Shewach Donna S. 
Published: August 2014

Abstract
Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which, if not repaired, augment sensitivity to subsequent ionizing radiation (IR). DNA mismatches were not required to elicit cytotoxicity but were necessary for radiosensitization to occur. We therefore hypothesized that RNAi mediated suppression of RR activity would also radiosensitize cells. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. To determine whether the suppression of either R2 or p53R2 produced a similar extent of radiosensitization compared to dFdCyd, two different shRNAs were used to suppress each subunit in wild-type p53 MCF7 breast carcinoma and A549 non-small cell lung carcinoma cells, producing a profound (≥ 90%) suppression of the target protein, R2 or p53R2, with little effect (≤20%) on expression of its homolog. shRNAs for both R2 and p53R2 produced effects similar to those observed with dFdCyd on dATP depletion (% control value) (MCF7: 66 ± 6% (shRNA) vs. 85 ± 10 % (dFdCyd); A549: 75 ± 20% (shRNA) vs. 80 ± 5% (dFdCyd), and cytotoxicity (MCF7: 35-60% (shRNA) vs. 40-50% (FdCyd); A549: 40-65% (shRNA) vs. 40-60% (dFdCyd)). When shRNA suppression was followed by IR, radiosensitivity was similar with the shRNAs (R2 and p53R2) vs. dFdCyd (MCF7: radiation enhancement ratio (RER)) = 1.45 ± 0.08 (shRNA) vs. 1.7 ± 0.14 (dFdCyd); A549: RER= 1.51 ± 0.09 (shRNA) vs. 1.55 ± 0.14(dFdCyd)), and a similar increase in DNA mismatches compared to untreated wild type cells (MCF7: ≥ 7-fold (dFdCyd) vs. 5-7 fold (shRNA); A549: ≥ 3 fold (dFdCyd) vs. 3-5 fold (shRNA)) was observed. Although p53R2 is thought to be induced primarily in response to DNA damage, its effects on dATP depletion and DNA mismatches prior to IR suggest that it may play a previously unidentified role in normal maintenance of DNA replication. These results reinforce our prior finding that the mechanism of radiosensitization with dFdCyd is the decrease in dATP and not its incorporation into DNA or its cytotoxicity. Excellent radiosensitization with suppression of either R2 or p53R2, compared to dFdCyd, suggests that this targeted approach merits in vivo evaluation. Elevated expression of R2 is known to enhance the invasiveness of cancer cells, and increase resistance to dFdCyd, thus its suppression would further enhance anticancer activity in addition to its effect on radiosensitization.
Citation Format: Sheryl A. Flanagan, Kristin Cooper, Sudha Mannava, Mikhail Nikiforov, Donna S. Shewach. shRNA mediated suppression of either of the small subunits of ribonucleotide reductase, R2 and p53R2, elicits a robust increase in sensitivity to ionizing radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2013-445