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.

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-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 (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 gammaH2AX 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 Thr2609 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.

Induction and repair of radiation-induced DNA double-strand breaks in human fibroblasts are not affected by terminal differentiation.

It was studied for human skin fibroblasts, whether the induction or repair of DNA double-strand breaks (dsb) depend on the differentiation status. These studies were performed (a) with a fibroblast strain (HSF1) kept in progenitor state (mitotic fibroblasts, MF) or triggered to premature terminal differentiation (postmitotic fibrocytes, PMF) by exposure to mitomycin C or (b) with 20 fibroblast strains differing intrinsically in their differentiation status. The differentiation status was quantified by determining the fraction of postmitotic fibrocytes by light microscopy. DNA dsb were measured by constant-field gel electrophoresis, and the fraction of apoptotic cells by comet assay. MF and PMF cultures of HSF1 cells were irradiated with X-ray doses up to 160 Gy, and dsb were measured either immediately after irradiation or after a repair incubation of 4 or 24 h. There were a difference neither in the number of initial nor residual dsb. PMF cultures, however, showed a slightly higher number of dsb already present in non-irradiated cells, which was measured to result from a small fraction of 5% apoptotic cells. The 20 analysed fibroblast strains showed a substantial variation in the fraction of postmitotic fibrocytes (9-51%) as well as in the number of dsb remaining at 24 h after irradiation (1.9-4.9%), but there was no correlation between these two parameters. These data demonstrate that for fibroblasts the terminal differentiation has an effect neither on the induction nor the repair of radiation-induced dsb. This result indicates that the variation in dsb-repair capacity previously observed for fibroblast strains and which was considered to be the main cause for the variation in the cellular radiosensitivity, cannot be ascribed to differences in the differentiation status.

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.

For X-irradiated normal human fibroblasts, only half of cell inactivation results from chromosomal damage.

PURPOSE: To study the relationship between residual double-strand breaks (dsbs), chromosomal damage, and cell inactivation for X-irradiated normal human fibroblasts. METHODS AND MATERIALS: The experiments were performed with 12 normal human fibroblast strains and, for comparison, a fibroblast line from a LiFraumeni patient (LFS2800), a squamous cell carcinoma line (FaDu), and CHO cells. Cells were irradiated in plateau phase, which was followed by immediate or delayed (14 h) plating. Chromosomal damage was measured by metaphase technique and loss of proliferative capacity by colony-forming assay. The data obtained were compared with residual double-strand breaks measured previously (Dikomey et al. IJROBP 2000;46:481-490). RESULTS: For each fibroblast strain, the number of lethal chromosome aberrations (CAs) increased with dose, but with a substantial variation among the strains (coefficient of variation = 20%-26%). The number of lethal aberrations was significantly correlated with the number of residual dsbs measured for the same strain (r(2) = 0.71, p = 0.0006). The residual dsbs were assumed to represent both non- and also mis-rejoined dsbs. There was a significant correlation between lethal aberrations and cell survival, but only for delayed and not immediate plating (r(2) = 0.69, p < 0.0008 vs. r(2) = 0.19, p = 0.16). For delayed plating, the ratio between lethal events (LEs) and CAs amounted to LE:CA = 2.0 +/- 0.05:1, indicating that on average, only half of cell inactivation resulted from chromosomal damage. The other 50% was attributed to the p53-dependent permanent G1 arrest, because cells lacking in functional p53 (LFS2800, FaDu, CHO) showed a ratio of LE:CA = 1.01 +/- 0.02:1. CONCLUSION: On average, up to 50% of the inactivation of X-irradiated normal human fibroblasts is a result of lethal chromosome aberrations, whereas the rest is due to a p53-dependent process, probably permanent G1 arrest.

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.

Synergistic cytotoxic activity of treosulfan and gemcitabine in pancreatic cancer cell lines.

BACKGROUND: Treatment for advanced pancreatic cancer is still very unsatisfactory. Treosulfan is an alkylating agent used for conventional, as well as high-dose chemotherapy regimens, whereby plasma concentrations over 500 µg/ml can be achieved. We investigated the effects of treosulfan on pancreatic cancer cell lines. MATERIALS AND METHODS: Using Panc-1, MIA PaCa-2 and Capan-2 cell lines, we investigated the in vitro cytotoxicity of treosulfan-alone and in combination with gemcitabine, 5-fluorouracil or irradiation. RESULTS: Treosulfan was potently cytotoxic against all pancreatic cancer cell lines at all concentrations (1-100 µg/ml). Combination of treosulfan and gemcitabine revealed strong synergistic effects independent of the sequence of drug administration. Similarly, synergism was observed with irradiation. Combination of treosulfan and 5-fluorouracil revealed antagonism. CONCLUSION: Treosulfan effectively kills pancreatic carcinoma cells in vitro and has synergistic activity in combination with gemcitabine and irradiation. These results warrant further investigation of treosulfan in the treatment of pancreatic cancer.