Cellular Radiobiology

Relationships between EGFR-initiated signalling, DNA double-strand breaks rejoining and survival in X-irradiated human glioma M059 cells

We investigated the effect of signalling inhibition on survival and double strand break (DSB) rejoining in cells differing in sensitivity to inhibitors, X-rays and bleomycin. Human glioma M059 cells, K (relatively radioresistant) and J (radiosensitive, defective in DSB rejoining for lack of DNA-dependent protein kinase catalytic subunit, DNA-PKcs) were pre-treated with signalling inhibitors: tyrphostin AG 1478, specific for epidermal growth factor receptor (EGFR) kinase or PD 98059, specific for kinase MEK 1,2 (mitogen-activated, extracellular signal-activated kinases 1 and 2). Subsequently, the cells were X-irradiated or treated with bleomycin. Cell survival was determined by clonogenicity test. DSB rejoining was monitored with the use of pulse field gel electrophoresis (PFGE).

We found that in X-irradiated M059 K cells EGFR kinase activity was necessary for efficient DSB rejoining and the kinase inhibitor, tyrphostin AG 1478, acted as radiosensitizer in the dose range that reduced cell survival to 0.7 – 0.8. Inhibition of EGFR kinase, however, did not decrease survival or affect DSB rejoining in DNA-PKcs-deficient M059 J cells. These results indicated that the decrease in cell survival was due to a disturbed DSB rejoining by the DNA-PK dependent system. In contrast, inhibition of MEK 1,2 kinase on EGFR downstream signalling pathway by PD 98059 did not affect DSB rejoining in either cell line and exerted a radioprotective effect.

Fig. 1. Survival curves for 2 M059 glioma cell lines.

Fig. 2. Rejoining of induced by X-rays DNA double strand breaks (DSB) in M059 K and M059 J cells, untreated (-T) or pre-treated (+T) with tyrphostine AG 1478, inhibitor of tyrosine kinase activity of EGFR.

Fig. 3.Accumulation of EGFR in nuclei of M059 K and M059 J cells, untreated (-T) or pre-treated (+T) with tyrphostine AG 1478 (T). Equitoxic inhibitor concentrations were applied: 4.5 μM (M059 K) and 9 μM (M059 J). Mean values +standard deviations are shown from 2 experiments.