Ingo Brammer

Blockage of Epidermal Growth Factor Receptor-Phosphatidylinositol 3-Kinase-AKT Signaling Increases Radiosensitivity of K-RAS Mutated Human Tumor Cells In vitro by Affecting DNA Repair

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 double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RASmt) cells. Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RASmt (A549) and K-RASwt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of γH2AX foci and frequency of micronuclei. Results: BIBX reduced clonogenic survival of K-RASmt-A549 cells, but not of K-RASwt-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-RASmt-A549 and MDA-MB-231 cells, but not in K-RASwt-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RASmt nor in K-RASwt 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.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair

PURPOSE Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. MATERIALS & METHODS Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. RESULTS Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. CONCLUSIONS The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

Molecular mechanisms of individual radiosensitivity studied in normal diploid human fibroblasts

The molecular mechanisms of individual radiosensitivity were studied in normal diploid human fibroblasts. For fibroblasts irradiated with X-rays in G1-phase the individual radiosensitivity was shown to be correlated with the extent of double-strand break (dsb) repair. The number of residual dsbs (including both non- and mis-rejoined dsbs) varied between 2 and 5% of the initial number induced and was low for resistant and high for sensitive strains. In the G1-phase dsbs are considered to be mostly repaired via the non-homologous end-joining pathway (NHEJ). However, so far none of the parameters tested for this pathway was found to be correlated with the number of residual dsbs. The parameters tested were mRNA expression, protein level and localisation and activity of the DNA-PK, which is the central complex of NHEJ. The dsb-repair capacity is also not regulated by the differentiation status, which varies substantially among fibroblast strains, whereas there is some indication that dsb repair might depend on the chromatin structure, with more efficient repair in cells with condensed DNA. Residual dsbs are converted into lethal chromosome aberrations finally leading to the loss of clonogenic activity, when cells pass through mitosis. Beside this so-called mitotic death, X-irradiated human fibroblasts are also inactivated via the TP53-dependent permanent G1-arrest, while apoptosis appears to be not important. On average, mitotic death and G1-arrest are equally effective, but there is a broad variation from one strain to the other, with a negative correlation between these two pathways. Fibroblast strains exhibiting only a moderate G1-arrest showed a high number of lethal aberrations and vice versa. This result points to a common regulator of both G1-arrest and dsb repair, which is presently under investigation.

Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest.

PURPOSE How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. MATERIALS AND METHODS We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. RESULTS When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. CONCLUSION For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation.