In situ Biological Dose Mapping Estimates the Radiation Burden Delivered to ‘Spared’ Tissue between Synchrotron X-Ray Microbeam Radiotherapy Tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to ‘spared’ tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30–40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells.</p

Biological in-vivo measurement of dose distribution in patients' lymphocytes by gamma-H2AX immunofluorescence staining: 3D conformal- vs. step-and-shoot IMRT of the prostate gland

<p>Abstract</p> <p>Background</p> <p>Different radiation-techniques in treating local staged prostate cancer differ in their dose- distribution. Physical phantom measurements indicate that for 3D, less healthy tissue is exposed to a relatively higher dose compared to SSIMRT. The purpose is to substantiate a dose distribution in lymphocytes <it>in-vivo </it>and to discuss the possibility of comparing it to the physical model of total body dose distribution.</p> <p>Methods</p> <p>For each technique (3D and SSIMRT), blood was taken from 20 patients before and 10 min after their first fraction of radiotherapy. The isolated leukocytes were fixed 2 hours after radiation. DNA double-strand breaks (DSB) in lymphocytes' nuclei were stained immunocytochemically using the gamma-H2AX protein. Gamma-H2AX foci inside each nucleus were counted in 300 irradiated as well as 50 non-irradiated lymphocytes per patient. In addition, lymphocytes of 5 volunteer subjects were irradiated externally at different doses and processed under same conditions as the patients' lymphocytes in order to generate a calibration-line. This calibration-line assigns dose-value to mean number of gamma-H2AX foci/ nucleus. So the dose distributions in patients' lymphocytes were determined regarding to the gamma-H2AX foci distribution. With this information a cumulative dose-lymphocyte-histogram (DLH) was generated. Visualized distribution of gamma-H2AX foci, correspondingly dose per nucleus, was compared to the technical dose-volume-histogram (DVH), related to the whole body-volume.</p> <p>Results</p> <p>Measured <it>in-vivo </it>(DLH) and according to the physical treatment-planning (DVH), more lymphocytes resulted with low-dose exposure (< 20% of the applied dose) and significantly fewer lymphocytes with middle-dose exposure (30%-60%) during Step-and-Shoot-IMRT, compared to conventional 3D conformal radiotherapy. The high-dose exposure (> 80%) was equal in both radiation techniques. The mean number of gamma-H2AX foci per lymphocyte was 0.49 (3D) and 0.47 (SSIMRT) without significant difference.</p> <p>Conclusions</p> <p><it>In-vivo </it>measurement of the dose distribution within patients' lymphocytes can be performed by detecting gamma-H2AX foci. In case of 3D and SSIMRT, the results of this method correlate with the physical calculated total body dose-distribution, but cannot be interpreted unrestrictedly due to the blood circulation. One possible application of the present method could be in radiation-protection for <it>in-vivo </it>dose estimation after accidental exposure to radiation.</p

Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

Copyright @ 2012 Nature Publishing GroupThis article has been made available through the Brunel Open Access Publishing Fund.Background: The objective of this study was to determine the molecular mechanism(s) responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double strand break (DSB) repair in cells. Quantitative-PCR (Q-PCR) established the expression levels of key DNA DSB repair proteins. Imaging flow cytometry using Annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Over-expression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.This work was supported in part by The Vidal Sassoon Foundation USA. This article is made available through the Brunel Open Access Publishing Fund

Comparison of established and emerging biodosimetry assays

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging &gamma;-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the &gamma;-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and &gamma;-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses &ge;1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools

Correlation between the radiation responses of fibroblasts cultured from individual patients and the risk of late reaction after breast radiotherapy.

Late normal tissue toxicity varies widely between patients and limits breast radiotherapy dose. Here we aimed to determine its relationship to DNA damage responses of fibroblast cultures from individual patients. Thirty-five breast cancer patients, with minimal or marked breast changes after breast-conserving therapy consented to receive a 4 Gy test irradiation to a small skin field of the left buttock and have punch biopsies taken from irradiated and unirradiated skin. Early-passage fibroblast cultures were established by outgrowth and irradiated in vitro with 0 or 4 Gy. 53BP1 foci, p53 and p21/CDKN1A were detected by immunofluorescence microscopy. Residual 53BP1 foci counts 24 h after in vitro irradiation were significantly higher in fibroblasts from RT-sensitive versus RT-resistant patients. Furthermore, significantly larger fractions of p53- but not p21/CDKN1A-positive fibroblasts were found in cultures from RT-sensitive patients without in vitro irradiation, and 2 h and 6 d post-irradiation. Exploratory analysis showed a stronger p53 response 2 h after irradiation of fibroblasts established from patients with severe reaction. These results associate the radiation response of fibroblasts with late reaction of the breast after RT and suggest a correlation with severity

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability