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Afterloading brachytherapy is conducted by the stepwise movement of a radioactive source through surgically implanted applicator tubes where at predefined dwell positions calculated dwell times optimize spatial dose delivery with respect to a planned dose level. The temporal exposure pattern exhibits drastic fluctuations in dose rate at a given coordinate and within a single treatment session because of the discontinuous and repeated source movement into the target volume. This could potentially affect biological response. Therefore, mammalian cells were exposed as monolayers to a high dose rate 192Ir source by utilizing a dedicated irradiation device where the distance between a planar array of radioactive source positions and the plane of the cell monolayer could be varied from 2.5 mm to 40 mm, thus varying dose rate pattern for any chosen total dose. The Gammamed IIi afterloading system equipped with a nominal 370 GBq (10 Ci) 192-Ir source was used to irradiate V79 Chinese hamster lung fibroblasts from both confluent and from exponential growth phase with dose up to 12 Gy (at room temperature, total exposure not exceeding 1 h). For comparison, V79 cells were also exposed to 6 MV x-rays from a clinical linear accelerator (dose rate of 2.5 Gy min−1). As biological endpoint, cell survival was determined by standard colony forming assay. Dose measurements were conducted with a diamond detector (sensitive area 7.3 mm2), calibrated by means of 60Co radiation. Additionally, dose delivery was simulated by Monte Carlo calculations using the EGSnrc code system. The calculated secondary electron fluence spectra at the cell location did not indicate a significant change of radiation quality (i.e. higher linear energy transfer) at the lower distances. Clonogenic cell survival curves obtained after brachytherapy exhibited an altered biological response compared to x-rays which was characterized by a significant reduction of the survival curve shoulder when dose rate fluctuations were high. Therefore, also for the time scale of the present investigation, cellular effects of radiation are not invariant to the temporal pattern in dose rate. We propose that with high dose rate variation the cells activate less efficiently their DNA damage response than after continuous irradiation.

Densely ionizing charged particle irradiation offers physical as well as biological advantages compared with photon irradiation. Radiobiological data for the combination of such particle irradiation (i.e. therapeutic carbon ions) with commonly used chemotherapeutics are still limited. Recent in vitro results indicate a general prevalence of additive cytotoxic effects in combined treatments, but an extension of established multimodal treatment regimens with photons to the inclusion of particle therapy needs to evaluate possible peculiarities of using high linear energy transfer (LET) radiation. The present study investigates the effect of combined radiochemotherapy using gemcitabine and high-LET irradiation with therapeutic carbon ions. In particular, the earlier observation of S-phase specific radiosensitization with photon irradiation should be evaluated with carbon ions. In the absence of the drug gemcitabine, carbon ion irradiation produced the typical survival behavior seen with X-rays-increased relative biological efficiency, and depletion of the survival curve's shoulder. By means of serum deprivation and subsequent replenishment, ∼70% S-phase content of the cell population was achieved, and such preparations showed radioresistance in both treatment arms-,photon and carbon ion irradiation. Combined modality treatment with gemcitabine caused significant reduction of clonogenic survival especially for the S-phase cells. WIDR cells exhibited S-phase-specific radioresistance with high-LET irradiation, although this was less pronounced than for X-ray exposure. The combined treatment with therapeutic carbon ions and gemcitabine caused the resistance phenomenon to disappear phenotypically.

Purpose: To present the first direct experimental in vitro comparison of the biological effectiveness of rangeequivalent protons and carbon ion beams for Chinese hamsterovary cells exposed in a three-dimensional phantom using a pencil beam scanning technique and to compare the experimental data with a novel biophysical model. Methodsand Materials:Cell survivalwasmeasuredinthe phantomafter irradiation withtwo opposingfields,thus mimicking the typical patient treatment scenario. The novel biophysical model represents a substantial extension of the local effect model, previously used for treatment planning in carbon ion therapy for more than 400 patients, and potentially can be used to predict effectiveness of all ion species relevant for radiotherapy. A key feature of the new approach is the more sophisticated consideration of spatially correlated damage induced by ion irradiation. Results: The experimental data obtained for Chinese hamster ovary cells clearly demonstrate that higher cell killing is achieved in the target region with carbon ions as compared with protons when the effects in the entrance channel are comparable. The model predictions demonstrate agreement with these experimental data and with data obtained with helium ions under similar conditions. Good agreement is also achieved with relative biological effectiveness values reported in the literature for other cell lines for monoenergetic proton, helium, and carbon ions. Conclusion: Both the experimental data and the new modeling approach are supportive of the advantages of carbon ions as compared with protons for treatment-like field configurations. Because the model predicts the effectiveness for several ion species with similar accuracy, it represents a powerful tool for further optimization and utilization of the potential of ion beams in tumor therapy. 2010 Elsevier Inc. Ion beam therapy, Relative biological effectiveness (RBE), Biophysical model, Treatment planning.

The repair of radiation-induced DNA double-strand breaks (DSBs) is frequently investigated by measuring the time-dependent decrease in the fraction of fragmented DNA that is able to enter electrophoresis gels. When transformed into equivalent doses without repair, such measurements are thought to reflect the removal of DSBs, and they typically exhibit a fast initial component and a decreasing rate at longer repair intervals. This formalism, however, assumes that the spatial distribution of unrejoined breakage resembles the pattern of induction of DSBs. While the size distributions for initial fragmentation, such as that resolved by conventional pulsed-field gel electrophoresis (PFGE) (between about 10(5) and 10(7) bp), are well known to agree with the prediction of random breakage, no data are available from studies explicitly testing this relationship for residual breakage. Therefore, Chinese hamster V79 cells and MeWo (human melanoma) cells were irradiated with different doses (10-100 Gy) or were incubated for repair for up to 4 h after a single dose of 100 Gy (V79) or 90 Gy (MeWo) before being subjected to PFGE. Fragment size distributions were calculated by convolution of the PFGE profiles with an appropriately generated size calibration function. The results clearly demonstrate an over-representation of smaller fragments (below about 2-3 Mbp) compared to the prediction of randomness for residual breakage. In consequence, the time-dependent decrease of dose-equivalent values calculated from data on the fraction released may not directly reflect DSB rejoining rates. The present findings are compatible with an earlier suggestion of slow rejoining of breaks which have been induced as multiple breaks (two or more) in large chromosomal loops, thus also predicting an increase of the slowly rejoining DSB fraction with increasing dose.

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.

Purpose Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)–activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKCα/β II was assessed by western blotting. Results No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than α/β II cannot be ruled out.

Purpose: Pemetrexed disodium (Alimta, multitargeted antifolate, LY231514; Eli Lilly and Co., Indianapolis, Indiana) (“pemetrexed”) is a new folate antimetabolite with significant antitumor activity. Different from classic antifolates, pemetrexed inhibits several key enzymes of thymidylate and purine synthesis, but a radiosensitizing potential may also be presumed. Therefore, the interaction of pemetrexed and ionizing radiation was studied for in vitro clonogenic survival of different human tumor cell lines. Methods and materials: Human colon (Widr), breast (MCF-7), cervix (Hela), and lung (LXI) carcinoma cells from log-phase cultures were exposed to pemetrexed (2 h) in combination with different radiation doses given 1 h before pemetrexed washout (all cell lines) or at different points of time before or after pemetrexed addition (Widr). Survival curves were analyzed according to the linear-quadratic (LQ) model, and mean inactivation doses (MID) and radiation enhancement ratios were calculated from the survival curve parameters. Cell-cycle progression of serum-stimulated and pemetrexed- or mock-treated Widr cells was monitored by flow cytometry. Results: Radiosensitization was found for all cell lines at moderately toxic pemetrexed exposures (0.05–0.3 μg/ml [106–636 nM]), but this was cell-type dependent and was most pronounced at roughly isotoxic concentrations, for the least pemetrexed-sensitive Widr cells. Enhancement ratios ranged from about 1.2 (MCF-7 and Hela) to 1.8 (Widr), with a tendency to increase with pemetrexed concentration. Little, if any, change of radiosensitization was observed (Widr) when the time of irradiation was varied from 4 h before to 10 h after the beginning of pemetrexed treatment. Cell-cycle progression of serum-stimulated Widr cells was only marginally affected by pemetrexed. Conclusions: Pemetrexed enhances radiation-induced cell inactivation at moderately toxic exposures and over many hours after drug removal. This effect is not due to disturbed cell-cycle progression, but likely involves an interaction of pemetrexed with long-lived (>4 h) cellular radiation damage and needs to be considered when introducing a combined clinical application.

This work aimed to study the effects of low dose irradiation in order to detect possible deviations from the linear-quadratic dose dependence of survival after charged particle irradiation. Survival of V79 cells at low doses was studied using a semi-automated cell detection system similar to the DMIPS system. With such a technique, the precision of survival measurements is improved by avoiding the stochastic uncertainties of the conventional dilution assay. A significant hypersensitivity at low doses (0.1 Gy) has been detected for 100 MeV/u carbon ions, whereas for higher doses (up to 0.5 Gy) a transition to a plateau-like region is observed, which is consistent with the assumption of induced resistance. These results are apparently in contrast to earlier studies using high-LET radiation like, e.g. neutrons or negative pions, where the effect of induced resistance has been reported to be at least significantly reduced for single exposure experiments.