Recent Advances in the Design of Anticancer Chemotherapy

Weber, George

doi:10.1159/000225492

Cited by 6 publications

(1 citation statement)

References 10 publications

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“…Early studies have demonstrated a positive correlation between the enzymatic activity of RNR and the rate of proliferation of transplanted Novikoff carcinomas (11,12). Thus, because of its critical role in the de novo synthesis of DNA, RNR is considered to be a potential target for the development of cancer chemotherapeutic agents.…”

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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

Lin

Belcourt

Cory

et al. 2004

Journal of Biological Chemistry

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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. Ribonucleotide reductase (RNR)1 catalyzes a rate-limiting reaction in which ribonucleoside diphosphates are converted to their corresponding deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis and repair (1). In mammalian cells, the catalytically active RNR is considered to be an ␣ 2 ␤ 2 heterotetramer consisting of two large R1 subunits and two small R2 subunits. To maintain a balanced size of dNTP pools (2), the enzymatic activity of RNR is tightly regulated by the binding of nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) to allosteric sites in the R1 subunit (1). In proliferating cells, the level of the R2 protein is low in the G 1 phase of the cell cycle but accumulates and reaches maximal levels during the S phase, followed by degradation when passing through the G 2 /M phase (3-6). Thus, the activity of RNR is additionally controlled by cell cycle-specific availability of the R2 subunit, whereas the R1 protein level remains relatively constant throughout the cell cycle (3, 4). When cells undergo G 1 arrest following DNA damage, the supply of dNTPs for DNA repair is, in turn, provided by transcriptional activation of the recently identified R2 homologue, p53R2 (7, 8). Thus, DNA damage causes p53-depend...

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confidence: 99%