Evidence for Recombinant GRP78, CALR, PDIA3 and GPI as Mediators of Genetic Instability in Human CD34+ Cells.

Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4−5-fold in response to any individual of the four factors, and centrosome anomalies by 3−4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks.

Intracellular Delivery of Doxorubicin by Iron Oxide-Based Nano-Constructs Increases Clonogenic Inactivation of Ionizing Radiation in HeLa Cells.

In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONPCO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONPCO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONPCO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMFSF0.1 = 1.13 ± 0.05 (p = 0.01)). IONPDOX did enhance the cytotoxicity of 6 MV X-rays (DMFSF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONPCO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONPDOX induced radiosensitization compared to IONPCO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Multi-centre technical evaluation of the radiation-induced lymphocyte apoptosis assay as a predictive test for radiotherapy toxicity.

Predicting which patients will develop adverse reactions to radiotherapy is important for personalised treatment. Prediction will require an algorithm or nomogram combining clinical and biological data. The radiation-induced lymphocyte apoptosis (RILA) assay is the leading candidate as a biological predictor of radiotherapy toxicity. In this study we tested the potential of the assay for standardisation and use in multiple testing laboratories. The assay was standardised and reproducibility determined using samples from healthy volunteers assayed concurrently in three laboratories in Leicester (UK), Mannheim (Germany) and Montpellier (France). RILA assays were performed on samples taken prior to radiotherapy from 1319 cancer patients enrolled in the REQUITE project at multiple centres. The patients were being treated for breast (n = 753), prostate (n = 506) or lung (n = 60) cancer. Inter-laboratory comparisons identified several factors affecting results: storage time, incubation periods and type of foetal calf serum. Following standardisation, there was no significant difference in results between the centres. Significant differences were seen in RILA scores between cancer types (prostate > breast > lung), by smoking status (non-smokers > smokers) and co-morbidity with rheumatoid arthritis (arthritics > non-arthritics). An analysis of acute radiotherapy toxicity showed as expected that RILA assay does not predict most end-points, but unexpectedly did predict acute breast pain. This result may elucidate the mechanism by which the RILA assay predicts late radiotherapy toxicity. The work shows clinical trials involving multiple laboratory measurement of the RILA assay are feasible and the need to account for tumour type and other variables when applying to predictive models.

In vivo micro-CT imaging of untreated and irradiated orthotopic glioblastoma xenografts in mice: capabilities, limitations and a comparison with bioluminescence imaging.

Small animal imaging is of increasing relevance in biomedical research. Studies systematically assessing the diagnostic accuracy of contrast-enhanced in vivo micro-CT of orthotopic glioma xenografts in mice do not exist. NOD/SCID/γc(-/-) mice (n = 27) underwent intracerebral implantation of 2.5 × 10(6) GFP-Luciferase-transduced U87MG cells. Mice underwent bioluminescence imaging (BLI) to detect tumor growth and afterwards repeated contrast-enhanced (300 µl Iomeprol i.v.) micro-CT imaging (80 kV, 75 µAs, 360° rotation, 1,000 projections, 33 s scan time, resolution 40 × 40 × 53 µm, 0.5 Gy/scan). Presence of tumors, tumor diameter and tumor volume in micro-CT were rated by two independent readers. Results were compared with histological analyses. Six mice with tumors confirmed by micro-CT received fractionated irradiation (3 × 5 Gy every other day) using the micro-CT (5 mm pencil beam geometry). Repeated micro-CT scans were tolerated well. Tumor engraftment rate was 74 % (n = 20). In micro-CT, mean tumor volume was 30 ± 33 mm(3), and the smallest detectable tumor measured 360 × 620 µm. The inter-rater agreement (n = 51 micro-CT scans) for the item tumor yes/no was excellent (Spearman-Rho = 0.862, p < 0.001). Sensitivity and specificity of micro-CT were 0.95 and 0.71, respectively (PPV = 0.91, NPV = 0.83). BLI on day 21 after tumor implantation had a sensitivity and specificity of 0.90 and 1.0, respectively (PPV = 1.0, NPV = 0.5). Maximum tumor diameter and volume in micro-CT and histology correlated excellently (tumor diameter: 0.929, p < 0.001; tumor volume: 0.969, p < 0.001, n = 17). Irradiated animals showed a large central tumor necrosis. Longitudinal contrast enhanced micro-CT imaging of brain tumor growth in live mice is feasible at high sensitivity levels and with excellent inter-rater agreement and allows visualization of radiation effects.