Deoxynucleoside Salvage Facilitates DNA Repair During Ribonucleotide Reductase Blockade in Human Cervical Cancers

Abstract

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.

Association of the Genetic Polymorphisms RRM1 −756T>C and −269C>A With Cervical Neoplasia

“…In another study, RRM1 expression increased in untreated cervical cancer tissues (Kunos et al, 2012). In a number of studies, cervical cancer cells promoted 2 0 -deoxyribonucleoside diphosphate production through RNR overactivity in order to repair radiochemotherapy-induced cancer cell damage and evade cancer cell death after radiochemotherapy (Kunos, Colussi, Pink, Radivoyevitch, & Oleinick, 2011;Kunos, Ferris, et al, 2011;Kunos et al, 2010). Moreover, RRM1 mRNA expression levels in diagnostic leukemic blasts were significantly associated with the promoter SNPs of RRM1 À756T>C (rs11030918) and À269C>A (rs12806698) among patients with acute myeloid leukemia (Cao et al, 2013).…”

“…Furthermore, supplementation with deoxynucleoside (0.50 mM) in a quantity 10-fold greater than that present in a standard serum-containing medium (0.05 mM) facilitates the repair of radiation-mediated DNA damage and reduces the radiochemosensitivity of human cervical cancer cells (Kunos, Ferris, Pyatka, Pink, & Radivoyevitch, 2011). DNA damage induced by radiation in a deoxynucleoside-free medium increases the deoxycytidine triphosphate concentration by increasing RNR activity (Kunos, Ferris, et al, 2011). Moreover, inhibiting RNR activity by using 3-aminopyridine-2-carboxaldehyde thiosemicarbazone enhances chemoradiosensitivity by sustaining DNA damage in human cervical cancer cells (Kunos et al, 2010).…”

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].…”

“…Of the two choices, cells appear to use the de novo ribonucleotide reductase path predominantly [42]. As a putative gatekeeper for deoxyribonucleotide supply, ribonucleotide reductase has a twostep DNA damage response repertoire.…”

“…3 Shown is a linear accelerator mounted on a robotic arm manipulator. a Cervical, endometrial, and ovarian cancer cell survivals on a vertical logarithmic scale are plotted after single fraction 800-cGy irradiation, without perturbation of deoxyribonucleotide production (dark blue) or either with blockade of ribonucleotide reductase by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (5 μM, aqua blue) for de novo block, or with media-free of deoxynucleosides ([dN]00, green) for impaired salvage as before [42]. b Depicted are radiation doses in centigray for individual "pencil" beams delivered during a 35-min radiosurgery treatment session.…”