Ribonucleotide Reductase Inhibition Enhances Chemoradiosensitivity of Human Cervical Cancers

Abstract

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 cancer.

Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma

“…3-AP is now used in clinical trials for a variety of advanced-stage solid tumors. [53][54][55] Toxicities reported from the phase 1 trial were hypoxia, respiratory distress, and methemoglobulinemia, apparently due to iron chelation in the red blood cells of the patients. 56 Recently, Zhou et al have developed a novel potent RR inhibitor, COH29, by using structure-and mechanism-based approaches, which displays a broad antitumor potential.…”

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

“…Most experiments concerning p53R2's function have been carried out with transformed cell lines (9,(17)(18)(19), which are not suitable for addressing these questions. To investigate p53R2 in nontransformed cells, we recently examined in vitro the consequences of p53R2 inactivation with fibroblasts from a patient with a lethal homozygous missense mutation in the iron-binding center of p53R2 who had died at aged 3 mo with severe muscular mtDNA depletion (16,20).…”

Stereotactic body radiation therapy in head and neck, gynecologic, and pediatric malignancies

“…Gynecologic cancer cells mend damage done by ionizing radiation in 3 h through an integrated cascade of proteins, predominantly using ribonucleotide reductase for de novo production of or using thymidine kinase 1 for salvage of deoxyribonucleotides [39][40][41][42][43]. Of the two choices, cells appear to use the de novo ribonucleotide reductase path predominantly [42].…”