Modulating radiation resistance by inhibiting ribonucleotide reductase in cancers with virally or mutationally silenced p53 protein

Abstract

Therapeutic ionizing radiation damages DNA, increasing p53-regulated ribonucleotide reductase (RNR) activity required for de novo synthesis of the deoxyribonucleotide triphosphates used during DNA repair. This study investigated the pharmacological inhibition of RNR in cells of virally or mutationally silenced p53 cancer cell lines using 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine(R), NSC #663249), a chemotherapeutic radiosensitizer that equally inhibits RNR M2 and p53R2 small subunits. The effects of 3-AP on RNR inhibition and resulting radiosensitization were evaluated in cervical (CaSki, HeLa and C33-a) and colon (RKO, RKO-E6) cancer cells. 3-AP treatment significantly enhanced radiation-related cytotoxicity in cervical and colon cancer cells. 3-AP treatment significantly decreased RNR activity, caused prolonged radiation-induced DNA damage, and resulted in an extended G(1)/S-phase cell cycle arrest in all cell lines. Similar effects were observed in both RKO and RKO-E6 cells, suggesting a p53-independent mechanism of radiosensitization. We conclude that inhibition of ribonucleotide reductase by 3-AP enhances radiation-mediated cytotoxicity independent of p53 regulation by impairing repair processes that rely on deoxyribonucleotide production, thereby substantially increasing the radiation sensitivity of human cancers.

FIG. 1

3-AP was an effective radiosensitizer in CaSki, HeLa and C33-a cervical cancer cells. Panel A: Cells were treated with radiation or 3-AP for 6 h. Radiation-related (P = 0.83) and 3-AP-related (P = 1.00) cytotoxicities in the three cervical cancer cell lines were not significantly different. Panel B: Cells were treated with radiation followed by escalating doses of 3-AP for 6 h. A significant positive radiation + 3-AP interaction was observed for all three cervical cancer cell lines (P < 0.001). For 1 µM 3-AP-radiation, CaSki (P = 0.97), HeLa (P = 0.69), and C33-a (P = 0.39) cytotoxicity was similar to radiation alone. Significant enhancement of cytotoxicity was observed for 5 µM 3-AP + radiation (P < 0.001) and 10 µM 3-AP + radiation (P < 0.001) compared to radiation alone. Error bars are ±SD.

FIG. 2

Treatment with 3-AP significantly lowered dCTP pools and caused elongation of the G₁/S cell cycle arrest. Panel A: Cells were treated with radiation (4 Gy), 3-AP (5 µM × 6 h), or radiation-3-AP (4 Gy, 5 µM × 6 h). 3-AP treatment alone led to a significant reduction in dCTP levels at 6 h after treatment in CaSki (P < 0.001), HeLa (P < 0.001), and C33-a (P < 0.01) cells compared to untreated cells. Radiation + 3-AP also led to a significant reduction in dCTP levels at 6 h in CaSki (P < 0.001), HeLa (P < 0.001), and C33-a (P < 0.001) cells compared to radiation alone. Panel B: Cells were untreated or treated with radiation (6 Gy) and/or 3-AP (5, 10 µM × 6 h) and harvested at the indicated times. DNA content was analyzed by propidium staining and flow cytometry in triplicate. Using CaSki cells as an illustrative example, 3-AP resulted in protracted G₁/S-phase arrest 18 h after 3-AP or radiation + 3-AP treatment (P values are comparisons of treatment groups for each time indicated). Cell cycle G₁/S arrest was similar after 5 µM 3-AP or 10 µM3-AP without (6 h: P = 1.00, 18 h: P = 0.98) or with (6 h: P = 0.12, 18 h: P = 1.00) radiation.

FIG. 3

Panel A: Treatment with 3-AP prolonged G₁/S-phase cell cycle arrest. Cells were treated with radiation (10 Gy), 3-AP (5 µM × 6 h), or radiation + 3-AP (10 Gy, 5 µM × 6 h) and harvested at 18, 24 and 36 h for DNA content analysis by flow cytometry in triplicate. While radiation-treated cells showed a G₂/M arrest, cells treated with radiation + 3-AP showed a greater G₁/S-phase cell cycle arrest. Panel B: Cells were untreated or treated with radiation (10 Gy) and/or exposed to 6 h of 3-AP (5 µM) and harvested at 18 h after treatment. At 18 h, CaSki cell p53 was not detected due to the presence of the HPV-16 E6. C33-a cells expressed a mutant p53, detected by antibody, that was unchanged after radiation or 3-AP. The downstream effector protein p21 showed a moderate increase after irradiation in CaSki cells but was not detected in C33-a cells.

FIG. 4

3-AP treatment led to an accumulation of DNA damage after irradiation. Panel A: HeLa cells were treated as indicated with 3-AP (5 µM × 6 h) and/or radiation (6 Gy) and harvested 4 h later for γ-H2AX analysis by microscopy. Radiation + 3-AP-treated cells demonstrated an increased number of γ-H2AX foci compared to radiation alone, indicative of DNA double-strand breaks. Panel B: Cells were untreated (closed circles) or were treated with 3-AP (5 µM × 6 h, open circles), radiation (6 Gy, closed triangles), or radiation + 3-AP (6 Gy, 5 µM × 6 h, open triangles) and harvested 1 and 4 h afterward for γ-H2AX analysis by flow cytometry. At 4 h after treatment, there were significant sustained γ-H2AX foci in radiation + 3-AP-treated cells where ribonucleotide reductase activity had been inhibited compared to radiation alone (P < 0.004, each cell line). Error bars are ±SD.

FIG. 5

RKO and RKO-E6 cells demonstrated enhanced radiosensitivity when treated with the 3-AP. Panel A: Cells were treated with 3-AP and/or radiation and assessed at 6 h after treatment. 3-AP treatment led to a significant reduction in dCTP levels (P < 0.01 for RKO and RKO-E6 cells with 3-AP alone; P < 0.001 for RKO and RKO-E6 cells with radiation + 3-AP). Panel B: Cells were treated with 3-AP and radiation as indicated and harvested 0 and 4 h afterward for analysis of DNA content by flow cytometry (P values are comparisons of treatment groups for each time indicated). After 3-AP treatment, a G₁/S arrest was apparent at 4 h in RKO and RKO-E6 cells. Panel C: After radiation + 3-AP compared to radiation alone, there was significant sustained DNA damage as indicated by persistent γ-H2AX foci (P < 0.001, each cell line). Panel D: After radiation + 3-AP treatment, there was a significant reduction in the clonogenic survival of RKO and RKO-E6 cells (P < 0.001). Error bars are ±SD.