Ribonucleotide reductase inhibition enhances chemoradiosensitivity of human cervical cancers.
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 cancers.
Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA. charles.kunos@uhhospitals.org
Charles A
CA
Kunos
Tomas
T
Radivoyevitch
John
J
Pink
Song-Mao
SM
Chiu
Tammy
T
Stefan
James
J
Jacobberger
Timothy J
TJ
Kinsella
D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D011725:Pyridines / Q000494:pharmacology; D011836:Radiation Tolerance / Q000187:drug effects* / Q000528:radiation effects; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013882:Thiosemicarbazones / Q000494:pharmacology; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000473:pathology*
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D011725:Pyridines; D013882:Thiosemicarbazones; C078157:3-aminopyridine-2-carboxaldehyde thiosemicarbazone; C405947:RRM2B protein, human; D012264:Ribonucleotide Reductases; D002945:Cisplatin