An Analytical Method for Quantifying the Yields of DNA Double-Strand Breaks Coupled with Strand Breaks by γ-H2AX Focus Formation Assay Based on Track-Structure Simulation.

Complex DNA double-strand break (DSB), which is defined as a DSB coupled with additional strand breaks within 10 bp in this study, induced after ionizing radiation or X-rays, is recognized as fatal damage which can induce cell death with a certain probability. In general, a DSB site inside the nucleus of live cells can be experimentally detected using the γ-H2AX focus formation assay. DSB complexity is believed to be detected by analyzing the focus size using such an assay. However, the relationship between focus size and DSB complexity remains uncertain. In this study, using Monte Carlo (MC) track-structure simulation codes, i.e., an in-house WLTrack code and a Particle and Heavy Ion Transport code System (PHITS), we developed an analytical method for qualifying the DSB complexity induced by photon irradiation from the microscopic image of γ-H2AX foci. First, assuming that events (i.e., ionization and excitation) potentially induce DNA strand breaks, we scored the number of events in a water cube (5.03 × 5.03 × 5.03 nm3) along electron tracks. Second, we obtained the relationship between the number of events and the foci size experimentally measured by the γ-H2AX focus formation assay. Third, using this relationship, we evaluated the degree of DSB complexity induced after photon irradiation for various X-ray spectra using the foci size, and the experimental DSB complexity was compared to the results estimated by the well-verified DNA damage estimation model in the PHITS code. The number of events in a water cube was found to be proportional to foci size, suggesting that the number of events intrinsically related to DSB complexity at the DNA scale. The developed method was applicable to focus data measured for various X-ray spectral situations (i.e., diagnostic kV X-rays and therapeutic MV X-rays). This method would contribute to a precise understanding of the early biological impacts of photon irradiation by means of the γ-H2AX focus formation assay.

Chromosomal Location of Genes Differentially Expressed in Tumor Cells Surviving High-Dose X-ray Irradiation: A Preliminary Study on Radio-Fragile Sites.

Altered gene expression is a common feature of tumor cells after irradiation. Our previous study showed that this phenomenon is not only an acute response to cytotoxic stress, instead, it was persistently detected in tumor cells that survived 10 Gy irradiation (IR cells). The current understanding is that DNA double-strand breaks (DSBs) are recognized by the phosphorylation of histone H2AX (H2AX) and triggers the ataxia-telangiectasia mutated (ATM) protein or the ATM- and Rad3-related (ATR) pathway, which activate or inactivate the DNA repair or apoptotic or senescence related molecules and causes the expression of genes in many instances. However, because changes in gene expression persist after passaging in IR cells, it may be due to the different pathways from these transient intracellular signaling pathways caused by DSBs. We performed microarray analysis of 30,000 genes in radiation-surviving cells (H1299-IR and MCF7-IR) and found an interesting relation between altered genes and their chromosomal loci. These loci formed a cluster on the chromosome, especially on 1q21 and 6p21-p22 in both irradiated cell lines. These chromosome sites might be regarded as "radio-fragile" sites.

Inorganic polyphosphate enhances radio-sensitivity in a human non-small cell lung cancer cell line, H1299.

Inorganic polyphosphate is a linear polymer containing tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. Polyphosphate has been recognized as a potent anti-metastasis reagent. However, the molecular mechanism underlying polyphosphate action on cancer cells is poorly understood. In this study, we investigated the involvement of polyphosphate in radio-sensitivity using a human non-small cell lung cancer cell line, H1299. We found that polyphosphate treatment decreases cellular adenosine triphosphate levels, suggesting a disruption of energy metabolism. We also found that the induction of DNA double-strand breaks was enhanced in polyphosphate-treated cells after X-ray irradiation and colony formation assay revealed that cell survival decreased compared with that of the control groups. These findings suggest that polyphosphate is a promising radio-sensitizer for cancer cells. Therefore, we hypothesized that polyphosphate treatment disrupts adenosine triphosphate-mediated energy transfer for cellular survival and DNA repair, thereby reducing the cellular capability to resist X-ray irradiation.

Evaluation of Size Correction Factor for Size-specific Dose Estimates (SSDE) Calculation.

American Association of Physicists in Medicine (AAPM) Report No.204 recommends the size-specific dose estimates (SSDE), wherein SSDE=computed tomography dose index-volume (CTDIvol )×size correction factor (SCF), as an index of the CT dose to consider patient thickness. However, the study on SSDE has not been made yet for area detector CT (ADCT) device such as a 320-row CT scanner. The purpose of this study was to evaluate the SCF values for ADCT by means of a simulation technique to look into the differences in SCF values due to beam width. In the simulation, to construct the geometry of the Aquilion ONE X-ray CT system (120 kV), the dose ratio and the effective energies were measured in the cone angle and fan angle directions, and these were incorporated into the simulation code, Electron Gamma Shower Ver.5 (EGS5). By changing the thickness of a PMMA phantom from 8 cm to 40 cm, CTDIvol and SCF were determined. The SCF values for the beam widths in conventional and volume scans were calculated. The differences among the SCF values of conventional, volume scans, and AAPM were up to 23.0%. However, when SCF values were normalized in a phantom of 16 cm diameter, the error tended to decrease for the cases of thin body thickness, such as those of children. It was concluded that even if beam width and device are different, the SCF values recommended by AAPM are useful in clinical situations.

The impact of dose rate on responses of human lens epithelial cells to ionizing irradiation.

The knowledge on responses of human lens epithelial cells (HLECs) to ionizing radiation exposure is important to understand mechanisms of radiation cataracts that are of concern in the field of radiation protection and radiation therapy. However, biological effects in HLECs following protracted exposure have not yet fully been explored. Here, we investigated the temporal kinetics of γ-H2AX foci as a marker for DNA double-strand breaks (DSBs) and cell survival in HLECs after exposure to photon beams at various dose rates (i.e., 150 kVp X-rays at 1.82, 0.1, and 0.033 Gy/min, and 137Cs γ-rays at 0.00461 Gy/min (27.7 cGy/h) and 0.00081 Gy/min (4.9 cGy/h)), compared to those in human lung fibroblasts (WI-38). In parallel, we quantified the recovery for DSBs and cell survival using a biophysical model. The study revealed that HLECs have a lower DSB repair rate than WI-38 cells. There is no significant impact of dose rate on cell survival in both cell lines in the dose-rate range of 0.033-1.82 Gy/min. In contrast, the experimental residual γ-H2AX foci showed inverse dose rate effects (IDREs) compared to the model prediction, highlighting the importance of the IDREs in evaluating radiation effects on the ocular lens.

Energy spectrum measurement of scattered X-rays during IVR procedure.

With the increase of the number of interventional radiology (IVR) procedures, the occupational exposure of operators and medical staff has attracted keen attention. The energy of scattered radiation in medical clinical sites is important for estimating the biological effects of occupational exposure. Recent years have seen many reports on the dose of scattered radiation by IVR, but few on the energy spectrum. In this study, the energy spectrum of scattered X-rays was measured by using a cadmium telluride (CdTe) semiconductor detector during IVR on several neurosurgical and cardiovascular cases. The cumulated spectra in each case were compared. The spectra showed little changes among neurosurgical cases and relatively large changes among cardiovascular cases. This was assumed to be due to the change of X-ray tube voltage and tube angle was larger in cardiovascular cases. The resulting energy spectra will be essential for the assessment of detailed biological effects of occupational exposure.

Graphical representation of the effects on tumor and OAR for determining the appropriate fractionation regimen in radiation therapy planning.

PURPOSE: The authors propose a graphical representation of the relation between the effect on the tumor and the damage effect on an organ at risk (OAR) against the irradiation dose, as an aid for choosing an appropriate fractionation regimen. METHODS: The graphical relation is depicted by the radiation effect on the tumor E(1) versus that on an OAR E(0). By observing the features of the E(1) vs E(0) relation curve, i.e., convex or concave shape, one can judge whether multifractionation is better or not. This method is applied to the linear-quadratic model (with α and ß parameters) as an example. Further, the method is extended to the general case for nonuniform dose distribution to the OAR, which is frequently seen in clinical situations. RESULTS: The criterion for selecting multi- or hypofractionation is based on the relation between the dose for the OAR and the α∕ß ratio of the OAR to the tumor. It is also shown that the graphical relation enables us to estimate the final effect after multifractionated treatment by plotting a tangent line on the curve. CONCLUSIONS: The graphical representation method is of use for improving planning in radiotherapy by determining the effective fractionation scheme.

Inflammatory Signaling and DNA Damage Responses after Local Exposure to an Insoluble Radioactive Microparticle.

Cesium-bearing microparticles (Cs-BMPs) can reach the human respiratory system after inhalation, resulting in chronic local internal exposure. We previously investigated the spatial distribution of DNA damage induced in areas around a Cs-BMP; however, the biological impacts have not been fully clarified due to the limited amount of data. Here, we investigated the inflammatory signaling and DNA damage responses after local exposure to a Cs-BMP in vitro. We used two normal human lung cell lines, i.e., lung fibroblast cells (WI-38) and bronchial epithelial cells (HBEC3-KT). After 24 h exposure to a Cs-BMP, inflammation was evaluated by immunofluorescent staining for nuclear factor κB (NF-κB) p65 and cyclooxygenase 2 (COX-2). The number of DNA double-strand breaks (DSBs) was also detected by means of phospholylated histone H2AX (γ-H2AX) focus formation assay. Cs-BMP exposure significantly increased NF-κB p65 and COX-2 expressions, which were related to the number of γ-H2AX foci in the cell nuclei. Compared to the uniform (external) exposure to 137Cs γ-rays, NF-κB tended to be more activated in the cells proximal to the Cs-BMP, while both NF-κB p65 and COX-2 were significantly activated in the distal cells. Experiments with chemical inhibitors for NF-κB p65 and COX-2 suggested the involvement of such inflammatory responses both in the reduced radiosensitivity of the cells proximal to Cs-BMP and the enhanced radiosensitivity of the cells distal from Cs-BMP. The data show that local exposure to Cs-BMP leads to biological effects modified by the NF-κB pathway, suggesting that the radiation risk for Cs-BMP exposure can differ from that estimated based on conventional uniform exposure to normal tissues.

Impact of the Lorentz force on electron track structure and early DNA damage yields in magnetic resonance-guided radiotherapy.

Magnetic resonance-guided radiotherapy (MRgRT) has been developed and installed in recent decades for external radiotherapy in several clinical facilities. Lorentz forces modulate dose distribution by charged particles in MRgRT; however, the impact of Lorentz forces on low-energy electron track structure and early DNA damage induction remain unclear. In this study, we estimated features of electron track structure and biological effects in a static magnetic field (SMF) using a general-purpose Monte Carlo code, particle and heavy ion transport code system (PHITS) that enables us to simulate low-energy electrons down to 1 meV by track-structure mode. The macroscopic dose distributions by electrons above approximately 300 keV initial energy in liquid water are changed by both perpendicular and parallel SMFs against the incident direction, indicating that the Lorentz force plays an important role in calculating dose within tumours. Meanwhile, DNA damage estimation based on the spatial patterns of atomic interactions indicates that the initial yield of DNA double-strand breaks (DSBs) is independent of the SMF intensity. The DSB induction is predominantly attributed to the secondary electrons below a few tens of eV, of which energy deposition patterns are not considerably affected by the Lorentz force. Our simulation study suggests that treatment planning for MRgRT can be made with consideration of only changed dose distribution.

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability.

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (-) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

Radiation-induced cancer cell repopulation: a possible mechanism implied by experiments using transplantable mouse-derived sarcoma cell line.

PURPOSE: Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. MATERIALS AND METHODS: A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. RESULTS: cDNA analysis showed that cyclin-dependent kinase inhibitors, p16 and p57 were down-regulated; 14.8- and 12.0-fold, respectively for the tolerant clone. Matrix metalloproteinase 3 and 13 were up-regulated; 22.5- and 25.8-fold, respectively. Transplantation ratio was 100% (5/5) for the tolerant clone whereas it was 40% (2/5) for the parent. Under light microscope, the mean mitotic cell number was 4.0+/-3.9 for the parent, and 12.8+/-3.4 for the tolerant clone (p<0.01, Student's t-test). CONCLUSIONS: This study implies that repopulation is not a temporary reaction to irradiation. It is caused probably by "clonal" gene-expression changes, though it remains unknown whether the changes are attributable to tolerant cell selection or to gene mutation/modification.

4-Methylumbelliferone administration enhances radiosensitivity of human fibrosarcoma by intercellular communication.

Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy.

Oxygen enhancement ratios of cancer cells after exposure to intensity modulated x-ray fields: DNA damage and cell survival.

Hypoxic cancer cells within solid tumours show radio-resistance, leading to malignant progression in fractionated radiotherapy. When prescribing dose to tumours under heterogeneous oxygen pressure with intensity-modulated radiation fields, intercellular signalling could have an impact on radiosensitivity between in-field and out-of-field (OF) cells. However, the impact of hypoxia on radio-sensitivity under modulated radiation intensity remains to be fully clarified. Here, we investigate the impact of hypoxia on in-field and OF radio-sensitivities using two types of cancer cells, DU145 and H1299. Using a nBIONIX hypoxic culture kit and a shielding technique to irradiate 50% of a cell culture flask, oxygen enhancement ratios for double-strand breaks (DSB) and cell death endpoints were determined. Thesein vitromeasurements indicate that hypoxia impacts OF cells, although the hypoxic impacts on OF cells for cell survival were dose-dependent and smaller compared to those for in-field and uniformly irradiated cells. These decreased radio-sensitivities of OF cells were shown as a consistent tendency for both DSB and cell death endpoints, suggesting that radiation-induced intercellular communication is of importance in advanced radiotherapy dose-distributions such as with intensity-modulated radiotherapy.

A Model for Estimating Dose-Rate Effects on Cell-Killing of Human Melanoma after Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a type of radiation therapy for eradicating tumor cells through a 10B(n,α)7Li reaction in the presence of 10B in cancer cells. When delivering a high absorbed dose to cancer cells using BNCT, both the timeline of 10B concentrations and the relative long dose-delivery time compared to photon therapy must be considered. Changes in radiosensitivity during such a long dose-delivery time can reduce the probability of tumor control; however, such changes have not yet been evaluated. Here, we propose an improved integrated microdosimetric-kinetic model that accounts for changes in microdosimetric quantities and dose rates depending on the 10B concentration and investigate the cell recovery (dose-rate effects) of melanoma during BNCT irradiation. The integrated microdosimetric-kinetic model used in this study considers both sub-lethal damage repair and changes in microdosimetric quantities during irradiation. The model, coupled with the Monte Carlo track structure simulation code of the Particle and Heavy Ion Transport code System, shows good agreement with in vitro experimental data for acute exposure to 60Co γ-rays, thermal neutrons, and BNCT with 10B concentrations of 10 ppm. This indicates that microdosimetric quantities are important parameters for predicting dose-response curves for cell survival under BNCT irradiations. Furthermore, the model estimation at the endpoint of the mean activation dose exhibits a reduced impact of cell recovery during BNCT irradiations with high linear energy transfer (LET) compared to 60Co γ-rays irradiation with low LET. Throughout this study, we discuss the advantages of BNCT for enhancing the killing of cancer cells with a reduced dose-rate dependency. If the neutron spectrum and the timelines for drug and dose delivery are provided, the present model will make it possible to predict radiosensitivity for more realistic dose-delivery schemes in BNCT irradiations.

A theoretical cell-killing model to evaluate oxygen enhancement ratios at DNA damage and cell survival endpoints in radiation therapy.

Radio-resistance induced under low oxygen pressure plays an important role in malignant progression in fractionated radiotherapy. For the general approach to predict cell killing under hypoxia, cell-killing models (e.g. the Linear-Quadratic model) have to be fitted to in vitro experimental survival data for both normoxia and hypoxia to obtain the oxygen enhancement ratio (OER). In such a case, model parameters for every oxygen condition needs to be considered by model-fitting approaches. This is inefficient for fractionated irradiation planning. Here, we present an efficient model for fractionated radiotherapy the integrated microdosimetric-kinetic model including cell-cycle distribution and the OER at DNA double-strand break endpoint (OERDSB). The cell survival curves described by this model can reproduce the in vitro experimental survival data for both acute and chronic low oxygen concentrations. The OERDSB used for calculating cell survival agrees well with experimental DSB ratio of normoxia to hypoxia. The important parameters of the model are oxygen pressure and cell-cycle distribution, which enables us to predict cell survival probabilities under chronic hypoxia and chronic anoxia. This work provides biological effective dose (BED) under various oxygen conditions including its uncertainty, which can contribute to creating fractionated regimens for multi-fractionated radiotherapy. If the oxygen concentration in a tumor can be quantified by medical imaging, the present model will make it possible to estimate the cell-killing and BED under hypoxia in more realistic intravital situations.

Determination of appropriate conversion factors for calculating size-specific dose estimates based on X-ray CT scout images after miscentering correction.

In this study, we proposed and evaluated the validity of an optimized size-specific dose estimate, a widely used index of radiation dose in X-ray computed tomography (CT) examinations. Based on miscentering correction of scout images, we determined the appropriate conversion factors (CF) by using a phantom. Scans were conducted using a multi-detector CT system (Aquilion ONE, Canon Medical Systems). Four cylindrical phantoms were taken in the anteroposterior (AP) and axial directions to determine the relationship between pixel value and water-equivalent length (Lw). In the AP scout image, the pixel values at the selected slice positions were converted to Lw to calculate the water-equivalent diameter (Dw). The CF was derived from Dw and CF values before and after miscentering correction was calculated. Finally, the CF values were compared to those calculated from the axial image using the conventional methodology of the American Association of Physicists in Medicine. Before miscentering correction, the maximum difference between the CF values of the axial and scout images was 7.26%. However, after miscentering correction, the maximum difference was 1.34%. Validation using a whole-body phantom generally revealed low maximum differences between the CF from the axial image and the values from the miscentering-corrected scout images. These were 2.41% in the chest, 6.30% in the upper abdomen, 1.43% in the abdomen, and 2.45% in the pelvic region. Consequently, we concluded that our miscentering correction method for deriving the appropriate CF values based on scout images is advantageous.