Azidothymidine enhances fluorodeoxyuridine-mediated radiosensitization

Abstract

Purpose: To examine the role of DNA repair and altered thymidine analogues in altering the response to radiation during thymidine deprivation.

Methods and materials: Mismatch repair-deficient and -proficient cell lines HEC59 and HC-2.4 were treated with fluorodeoxyuridine (FUdR), azidothymidine (AZT), and irradiation either alone or in combination, and outcomes of clonogenic survival and cell-cycle distributions were determined.

Results: Survival outcomes for all treatments were similar for both cell lines, suggesting that hMSH2 does not significantly influence thymidine deprivation toxicity or radiosensitization. The chain-terminating thymidine analogue AZT increased the toxicity of FUdR and increased DNA fragmentation. The combination of FUdR and AZT afforded greater radiosensitization than either drug alone. Drug enhancement ratios, the degree of excess radiation-induced cell death in drug-treated cultures compared with radiation alone for HEC59, were 1.2, 1.4, and 1.8 for AZT, FUdR, and the combination, respectively. Enhancement ratios for HC-2.4 were 1.3, 1.5, and 1.8 for AZT, FUdR, and the combination, respectively.

Conclusion: Azidothymidine, a chain-terminating thymidine analogue, can enhance the radiosensitizing affects of thymidine deprivation. Deoxyribonucleic acid strand breaks may play an important role in the mechanism of thymidine deprivation-induced radiosensitization.

Figure 1

Diagram of study design and treatment delivery.

Figure 2

Drug cytotoxicity dose response. A) Cells were plated and allowed to adhere for 24 hours then treated for 48 hours with varying doses of FUdR or control as described in Methods. After treatment, all cells were collected, washed, counted and replated to determine survival. B) Similar treatments were conducted with varying doses of AZT.

Figure 3

Combined treatment increases toxicity. Cells were treated with FUdR (30 micromolar), AZT (1 mM) or the combination as outlined in Figure 1 and in Methods. Clonogenic survival was determined.

Figure 4

Cell cycle changes resulting from drug treatment and recovery. HEC59 cells were exposed to 30 micromolar FUdR, 1 mM AZT or a combination. Samples of cells were collected after 48 hours of FUdR treatment alone, 24 hours of AZT treatment alone and concurrent treatment as shown in figure 1. Control cultures not treated with drug were also analyzed. Cells were collected, fixed and stained with propidium iodide as described in methods. HEC59+2 cultures were analyzed also with similar results. The scale for x- and y-axes are constant for all conditions shown. Parallel experiments in HEC59+2 gave similar results.

Figure 5

Radiation sensitivity of HEC59 and HEC59+2 cells is comparable. Cells were plated and allowed to attach for 24 hours. Plates were then exposed to varying doses of radiation as described in Methods. Plates were stained and colonies counted after 14 days.

Figure 6

Radiosensitization by thymidine analogs. A) Cells were treated with 30 micromolar FUdR for 48hours, irradiated. Treated cells were then collected, counted and replated. Relative survival was determined compared to no drug and plotted as shown. B) Cells were treated with 1 mM AZT for 24 hours then irradiated and treated as described for A. C) Cells were treated with 30 micromolar FUdR for 48 hours and 1 mM AZT concurrently during the second 24 hour period. Cells were treated as described above.