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.
Kunos
Charles A
CA
Department of Radiation Oncology, University Hospitals Case Medical Center and Case Western Reserve School of Medicine, Cleveland, Ohio 44106, USA. charles.kunos@uhhospitals.org
Radivoyevitch
Tomas
T
Pink
John
J
Chiu
Song-Mao
SM
Stefan
Tammy
T
Jacobberger
James
J
Kinsella
Timothy J
TJ
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