Purpose: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA doublestrand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS mt ) cells. Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS mt (A549) and K-RAS wt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser 139 ), ATM (Ser 1981 ), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr 2609 ) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gH2AX foci and frequency of micronuclei. Results: BIBX reduced clonogenic survival of K-RAS mt -A549 cells, but not of K-RAS wt -FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS mt -A549 and MDA-MB-231 cells, but not in K-RAS wt -FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr 2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS mt nor in K-RAS wt cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. Conclusion: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

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The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24h after irradiation can be used as an indicator of cellular radiosensitivity.

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The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7.5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3.8 +/- 1.4, 1.4 +/- 0.8 or 0 +/- 0.7 days for 7.5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4.7 +/- 1.8, 2.6 +/- 0.7 or 2.1 +/- 0.4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2.7 +/- 0.7 to 5.0 +/- 0.8 or 6.3 +/- 1.0 days for 7.5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3.7 +/- 0.2 days) was 3.1 +/- 0.1, 2.1 +/- 0.1 and 1.1 +/- 0.1 days for the three doses applied.(ABSTRACT TRUNCATED AT 400 WORDS)

The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.