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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.

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.

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.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.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.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.

Purpose: To investigate the relationship between DNA double-strand breaks (dsbs), cell killing, and fibrosis using skin fibroblasts derived from breast cancer patients who received postmastectomy radiotherapy. Methods and Materials: Experiments were performed with 12 lines of normal skin fibroblasts derived from recurrence-free breast cancer patients. Cells were irradiated in confluence and cell survival was determined either after immediate or delayed (14 h) plating using a colony-forming assay. Dsbs were measured by constant-field gel electrophoresis. The "excess risk of fibrosis" was previously scored by Johansen et al. ( IJRB 1994;66:407–412). Results: The 12 cell lines showed a typical spectrum of radiosensitivity. The mean value of surviving fraction after 3.5 Gy (SF3.5) was 0.063 for immediate and 0.174 for delayed plating with a coefficient of variation (CV) of 44 and 39%, respectively. There was also a broad variation in the extent of recovery from potentially lethal damage (RPLD), which was not correlated with the immediate sensitivity. The number of initial dsbs as well as the half-times of dsb repair showed little variation, whereas there were considerable differences in the number of residual dsbs (CV = 29%). The number of residual dsbs after 100 Gy was correlated significantly only with SF3.5 after delayed ( r 2 = 0.59; p = 0.006) but not after immediate plating ( r 2 = 0.21, p = 0.16). There was also no significant relationship between residual dsbs and the "excess risk of fibrosis" determined for the respective patients. Conclusion: It is shown that the number of residual dsbs measured in confluent human fibroblast lines can be used to predict the cellular radiosensitivity after delayed but not after immediate plating and also not to predict the excess risk of fibrosis of the respective breast cancer patients.

The effect of heat (43-45 degrees C) on the induction and the repair of DNA double-strand breaks (dsb) was studied in CHO cells after 60 Gy of X-rays using constant-field gel electrophoresis. Heat given prior to irradiation was found not to alter the number of dsb when measured immediately after irradiation. In non-heated cells, about 80% of all dsb were rapidly repaired with a half-time of 4 min, while 20% were repaired more slowly with T4 = 160 min. These kinetics were grossly altered by heat. Both the fast and the slow process were retarded. However, the main effect of heat was an increase in the number of slowly rejoined dsb. This increase was shown to result from the additional formation (up to 1.4-fold the initial number) of dsb early during the repair course. It is suggested that the additional dsb arose from base damage, the repair of which was unbalanced by heat. No evidence was found for apoptosis being involved in this process. The kinetics of the additional dsb was found to correlate with thermal radiosensitization.

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 γH2AX-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.

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.

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 (LF S 2800), 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 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.

To test the relationship between cell killing and non-repaired DNA strand breaks both in repair proficient and deficient cell lines.Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs1, xrs5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis.The nine cell lines showed a broad spectrum in radiosensitivity with SF2 values ranging from 0.018 to 0.58. The cell lines did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r2=0.29) but a fairly good correlation for double-strand breaks (r2=0.87). This correlation was found to hold both for repair proficient and deficient cell lines.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.