Reversibility of Inhibition of DNA Double Strand Break Repair by 2-Deoxy-d-Glucose in Ehrlich Ascites Tumour Cells

Summary

The glycolytic inhibitor 2-deoxy-d-glucose (2-DG), a potent therapeutic adjuvant in cancer radiotherapy, was tested for the reversibility of its inhibitory action on X-ray-induced DNA double strand break (dsb) repair. Cells were exposed to 40 Gy of X-rays and allowed to repair with or without 2-DG in suspension at 37°C. DNA dsb rejoining was measured by pulsed field gel electrophoresis. The fraction of ¹⁴C-thymidine activity released from the plug (FAR) during electrophoresis was used as a measure for the number of dsb present in the DNA. After certain time intervals 2-DG was withdrawn from the cells and the extent of reversal of inhibition of dsb rejoining was measured. Biphasic repair of dsb was generally observed, with a fast component extending up to about 60 min after irradiation and a much slower progression of repair thereafter. Different mathematical models are considered for a quantitative description of these two components. The experimental data strongly indicate that only one type of dsb is primarily induced by irradiation which can be repaired fast with a time constant of about 0·05 min⁻¹ (t_1/2 ∼ 13 min). In competition with this repair, other DNA dsb arise which are repaired slowly with a time constant of about 0·009 min⁻¹ (t_1/2 ∼ 77 min). The time constant for the transformation of fast reparable dsb into slowly repaired dsb is about 0·026 min⁻¹. Treatment with 2-DG inhibits the fast repair and, as a consequence, more DNA dsb are transformed into the type being repaired slowly. In competition with this slow repair, DNA dsb are fixed. Treatment with 2-DG also reduces slow repair processes and as a consequence the number of lesions being fixed is increased. Cell survival and ATP content of the cells showed a reversibility to the same extent as dsb repair, indicating the close relationship of these processes in living cells.