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 cancers.

FIG. 1

Blockade of ribonucleotide reduction by 3-AP perpetuates DNA damage and enhances cytotoxicity in CaSki cervical cancer cells. Panels a–h show γ-H2AX levels measured by flow cytometry in CaSki cells 6 h after treatment with radiation (IR, 6 Gy), cisplatin (5 μM), and/or 3-AP (5 μM). Panels i–p show γ-H2AX foci 6 h after treatment acquired by laser scanning imaging cytometry. Panels q–x show the percentage of cells with measurable γ-H2AX at 0, 1, 4 and 6 h after treatment. Panel y shows dNTP pools as measured by a DNA polymerase extension assay 6 h after treatment. Panel z depicts clonogenic survival of cells after ionizing radiation exposure (0–10 Gy) alone, in combination with 5 μM cisplatin, with 3-AP (5 μM) and with both cisplatin (5 μM) and 3-AP (5 μM). Panel zz depicts clonogenic survival of CaSki cells treated for 6 h with a range of 3-AP doses (0–10 μM) alone or in combination with cisplatin (5 μM) or cisplatin (5 μM) plus ionizing radiation (20 Gy). Error bars are ±SD.

FIG. 2

Blockade of ribonucleotide reduction by 3-AP perpetuates DNA damage and enhances cytotoxicity in C33-a cervical cancer cells. Panels a–h show γ-H2AX levels measured by flow cytometry in C33-a cells 6 h after treatment with radiation (IR, 6 Gy), cisplatin (5 μM), and/or 3-AP (5 μM). Panels i–p show γ-H2AX foci 6 h after treatment acquired by laser scanning imaging cytometry. Panels q–x show the percentage of cells with measurable γ-H2AX at 0, 1, 4 and 6 h after treatment. Panel y shows dNTP pools as measured by a DNA polymerase extension assay 6 h after treatment (*P < 0.001). Panel z depicts clonogenic survival of cells after ionizing radiation exposure (0–10 Gy) alone, in combination with 5 μM cisplatin, with 3-AP (5 μM) and with both cisplatin (5 μM) and 3-AP (5 μM). Panel zz depicts clonogenic survival of C33-a cells treated for 6 h with a range of 3-AP doses (0–10 μM) alone or in combination with cisplatin (5 μM) or cisplatin (5 μM) plus ionizing radiation (20 Gy). Error bars are ±SD.

FIG. 3

DNA damage is associated with increased p53R2 expression 6 h after radiation or cisplatin treatment. Panel A illustrates laser scanning imaging cytometry of G₁-phase CaSki cervical cancer cells 6 h after irradiation alone (2 Gy), after irradiation (2 Gy), 5 μM cisplatin plus 5 μM 3-AP treatment, and after no treatment. Nuclei are stained blue by DAPI, p53R2 stained yellow by rabbit anti-human p53R2·Alexa Fluor 555, p21 stained red by rabbit anti-human p21·Alexa Fluor 647, and DNA damage stained green by mouse anti-human FITC-conjugated γ-H2AX antibodies. Dot plot depicts p53R2 intensity by DNA content (bar 5 mean of radiation-treated cells, arrow 5 cell depicted above in panel A). Sustained DNA damage at 6 h is associated with increased p53R2. Similar molecular results are found in C33-a cells (data not shown). Panel B depicts laser scanning imaging cytometry intensity (circles) of γ-H2AX and p53R2 signal at 6 h after irradiation (2 Gy) and/or cisplatin (5 μM) and/or 3-AP (5 μM) as indicated. Triangles are mean intensity. A significant rise in p53R2 steady-state protein levels is observed after DNA damage. No change in p21 relative to control is detected (data not shown). Panel C illustrates that cell cycle distribution of exponentially growing cells has not been affected substantially 6 h after DNA damage. In panel D, C33-a cells were exposed to radiation (2 Gy) and/or cisplatin (5 μM) and/or 3-AP (5 μM) as indicated. Western blot (WB) antibodies show a moderate rise in γ-H2AX protein 6 h after DNA damage. Compensatory rises in 6-h steady-state levels of p53R2 are apparent after DNA damage. No p21 protein was detected by Western blotting 6 h after DNA damage in CaSki or C33-a cells (data not shown).