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J. Biol. Chem., Vol. 279, Issue 26, 27030-27038, June 25, 2004

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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^*

Z. Ping Lin, Michael F. Belcourt, Joseph G. Cory¶, and Alan C. Sartorelli||

From the Department of Pharmacology and Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520, Vion Pharmaceuticals Incorporated, New Haven, Connecticut 06511, and the ^¶Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27834

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.

Received for publication, February 25, 2004 , and in revised form, March 25, 2004.

^* 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.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4533; Fax: 203-737-2045; E-mail: alan.sartorelli{at}yale.edu.

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