Hypoxia is well known to limit curability of tumors by ionizing radiation. Here, we show that hypoxia treatment of tumor cells causes coexpression of heat shock protein 70 (Hsp70) and phosphatidylserine (PS) on the cell surface. Colocalization of Hsp70 and PS, as determined by confocal microscopy, also occurs when exogenous FITC-labeled Hsp70 protein is added to normoxic and hypoxic tumor cells. Moreover, the interaction of Hsp70 with PS was demonstrated in artificial unilamellar phosphatidylcholine/ phosphatidylserine (PC/PS) liposomes at the physiological ratio of 8/2. Indeed, the Hsp70-liposome interaction gradually increased with elevating PS molar ratios (8/2 > or = 7/3 < 5/5 < 4/6 < 3/7 < 2/8). In contrast, only a weak Hsp70 interaction was detected in phosphatidylcholine/phosphatidylglycerol (PC/PG) liposomes, thus demonstrating that the interaction was not a charge-related effect. The interaction of Hsp70 with surface PS significantly reduces clonogenic cell survival in normoxic (EC(50) of Hsp70=85 microg/ml) and hypoxic (EC(50) of Hsp70=55 microg/ml) tumor cells. The radiation-induced tumor cell killing was significantly enhanced by the addition of Hsp70 protein (50 microg/ml). Since apoptosis was not significantly enhanced in normoxic and hypoxic tumor cells by the addition of Hsp70, we hypothesize that the Hsp70 protein-induced reduction in clonogenic cell survival might be through necrosis rather than apoptosis.

The risk of a large-scale event leading to acute radiation exposure necessitates the development of high-throughput methods for providing rapid individual dose estimates. Our work addresses three goals, which align with the directive of the European Union's Realizing the European Network of Biodosimetry project (EU-RENB): 1. To examine the suitability of different gene expression platforms for biodosimetry purposes; 2. To perform this examination using blood samples collected from prostate cancer patients (in vivo) and from healthy donors (in vitro); and 3. To compare radiation-induced gene expression changes of the in vivo with in vitro blood samples. For the in vitro part of this study, EDTA-treated whole blood was irradiated immediately after venipuncture using single X-ray doses (1 Gy/min(-1) dose rate, 100 keV). Blood samples used to generate calibration curves as well as 10 coded (blinded) samples (0-4 Gy dose range) were incubated for 24 h in vitro, lysed and shipped on wet ice. For the in vivo part of the study PAXgene tubes were used and peripheral blood (2.5 ml) was collected from prostate cancer patients before and 24 h after the first fractionated 2 Gy dose of localized radiotherapy to the pelvis [linear accelerator (LINAC), 580 MU/min, exposure 1-1.5 min]. Assays were run in each laboratory according to locally established protocols using either microarray platforms (2 laboratories) or qRT-PCR (2 laboratories). Report times on dose estimates were documented. The mean absolute difference of estimated doses relative to the true doses (Gy) were calculated. Doses were also merged into binary categories reflecting aspects of clinical/diagnostic relevance. For the in vitro part of the study, the earliest report time on dose estimates was 7 h for qRT-PCR and 35 h for microarrays. Methodological variance of gene expression measurements (CV ≤10% for technical replicates) and interindividual variance (≤twofold for all genes) were low. Dose estimates based on one gene, ferredoxin reductase (FDXR), using qRT-PCR were as precise as dose estimates based on multiple genes using microarrays, but the precision decreased at doses ≥2 Gy. Binary dose categories comprising, for example, unexposed compared with exposed samples, could be completely discriminated with most of our methods. Exposed prostate cancer blood samples (n = 4) could be completely discriminated from unexposed blood samples (n = 4, P < 0.03, two-sided Fisher's exact test) without individual controls. This could be performed by introducing an in vitro-to-in vivo correction factor of FDXR, which varied among the laboratories. After that the in vitro-constructed calibration curves could be used for dose estimation of the in vivo exposed prostate cancer blood samples within an accuracy window of ±0.5 Gy in both contributing qRT-PCR laboratories. In conclusion, early and precise dose estimates can be performed, in particular at doses ≤2 Gy in vitro. Blood samples of prostate cancer patients exposed to 0.09-0.017 Gy could be completely discriminated from pre...

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 γ-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 γ-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 γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥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.

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.

Anthropometric factors, especially BMI and body fat percentage, were significantly associated with increased risk of PTC. Future studies of anthropometric factors and PTC that incorporate intermediate factors, including adiposity and hormone biomarkers, are essential to help clarify potential mechanisms of the relationship.

A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2-positive and -negative cases-a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation-induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P=0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n=33), two independent validation cohorts of PTCs (n=115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC.

In recent years, the significance of apoptosis as a process in cell loss from normal tissue and tumours has been critically reviewed. In addition, the general lack of a correlation between radiation or drug-induced apoptosis and cell survival responses (using the clonogenic assay) in tumour cells has been demonstrated. Several different reasons have been discussed by other authors. It is the purpose of this review to argue that there are many different forms of cell death (terminal differentiation, micronucleation, mitotic catastrophe or multinucleation) that, like apoptosis, are regulated by the cell. In this context, apoptosis was the first cell death mechanism associated with active involvement of the cell (signal transduction). Furthermore, a large variety of different in vitro and a few in vivo models published so far show that the form of cell death can shift from, for example, mitotic catastrophe to apoptosis. The shift appears to be a general principle and depends on the cell model examined, the stressor type and the stressor intensity. These considerations help to explain the absence of a simple link between apoptosis and clonogenicity and suggest how to overcome that limitation, which has implications for the significance of apoptosis where the diagnosis and prognosis of cancer are concerned.

The possibility of a large-scale acute radiation exposure necessitates the development of new methods that could provide rapid individual dose estimates with high sample throughput. The focus of the study was an intercomparison of laboratories’ dose-assessment performances using gene expression assays. Lithium-heparinized whole blood from one healthy donor was irradiated (240 kVp, 1 Gy/min) immediately after venipuncture at approximately 37°C using single X-ray doses. Blood samples to establish calibration curves (0.25–4 Gy) as well as 10 blinded test samples (0.1–6.4 Gy) were incubated for 24 h at 37°C supplemented with an equal volume of medium and 10% fetal calf serum. For quantitative reverse transcription polymerase chain reaction (qRT-PCR), samples were lysed, stored at −20°C and shipped on ice. For the Chemical Ligation Dependent Probe Amplification methodology (CLPA), aliquots were incubated in 2 ml CLPA reaction buffer (DxTerity), mixed and shipped at room temperature. Assays were run in each laboratory according to locally established protocols. The mean absolute difference (MAD) of estimated doses relative to the true doses (in Gy) was calculated. We also merged doses into binary categories reflecting aspects of clinical/diagnostic relevance and examined accuracy, sensitivity and specificity. The earliest reported time on dose estimates was <8 h. The standard deviation of technical replicate measurements in 75% of all measurements was below 11%. MAD values of 0.3–0.5 Gy and 0.8–1.3 Gy divided the laboratories contributions into two groups. These fourfold differences in accuracy could be primarily explained by unexpected variances of the housekeeping gene (P =0.0008) and performance differences in processing of calibration and blinded test samples by half of the contributing laboratories. Reported gene expression dose estimates aggregated into binary categories in general showed an accuracies and sensitivities of 93–100% and 76–100% for the groups, with low MAD and high MAD, respectively. In conclusion, gene expression-based dose estimates were reported quickly, and for laboratories with MAD between 0.3–0.5 Gy binary dose categories of clinical significance could be discriminated with an accuracy and sensitivity comparable to established cytogenetic assays.