Puma and Trail/Dr5 Pathways Control Radiation-Induced Apoptosis in Distinct Populations of Testicular Progenitors

Spermatogonia- stem cells and progenitors of adult spermatogenesis- are killed through a p53-regulated apoptotic process after γ-irradiation but the death effectors are still poorly characterized. Our data demonstrate that both intrinsic and extrinsic apoptotic pathways are involved, and especially that spermatogonia can be split into two main populations, according to apoptotic effectors. Following irradiation both Dr5 and Puma genes are upregulated in the α6-integrin-positive Side Population (SP) fraction, which is highly enriched in spermatogonia. Flow cytometric analysis confirms an increased number of Dr5-expressing SP cells, and Puma-β isoform accumulates in α6-integrin positive cellular extracts, enriched in spermatogonia. Trail−/− or Puma−/− spermatogonia display a reduced sensitivity to radiation-induced apoptosis. The TUNEL kinetics strongly suggest that the extrinsic and intrinsic pathways, via Trail/Dr5 and Puma respectively, could be engaged in distinct subpopulations of spermatogonia. Indeed flow cytometric studies show that Dr5 receptor is constitutively present on more than half of the undifferentiated progenitors (Kit− α6+ SP) and half of the differentiated ones (Kit+ α6+ SP). In addition after irradiation, Puma is not detected in the Dr5-positive cellular fraction isolated by immunomagnetic purification, while Puma is present in the Dr5-negative cell extracts. In conclusion, adult testicular progenitors are divided into distinct sub-populations by apoptotic effectors, independently of progenitor types (immature Kit-negative versus mature Kit-positive), underscoring differential radiosensitivities characterizing the stem cell/progenitors compartment

Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

<p>Abstract</p> <p>Background</p> <p>The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both <it>in vitro </it>and <it>in vivo</it>, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes.</p> <p>Methods</p> <p>Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent <it>in situ </it>labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test.</p> <p>Results</p> <p>Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, <it>RELB </it>was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells.</p> <p>Conclusion</p> <p>The molecular defects in the silencing of <it>RELB </it>involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic <it>RELB </it>silencing as a new marker of the progressive disease in males.</p

Gene expression signature discriminates sporadic from post-radiotherapy-induced thyroid tumors

Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors

Gold Nanoparticle Uptake in Tumor Cells: Quantification and Size Distribution by sp-ICPMS

Gold nanoparticles (AuNPs) are increasingly studied for cancer treatment purposes, as they can potentially improve both control and efficiency of the treatment. Intensive research is conducted in vitro on rodent and human cell lines to objectify the gain of combining AuNPs with cancer treatment and to understand their mechanisms of action. However, using nanoparticles in such studies requires thorough knowledge of their cellular uptake. In this study, we optimized single particle ICPMS (sp-ICPMS) analysis to qualify and quantify intracellular AuNP content after exposure of in vitro human breast cancer cell lines. To this aim, cells were treated with an alkaline digestion method with 5% TMAH, allowing the detection of gold with a yield of 97% on average. Results showed that under our experimental conditions, the AuNP size distribution appeared to be unchanged after internalization and that the uptake of particles depended on the cell line and on the exposure duration. Finally, the comparison of the particle numbers per cell with the estimates based on the gold masses showed excellent agreement, confirming the validity of the sp-ICPMS particle measurements in such complex samples

Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

Delayed cell death by mitotic catastrophe is a frequent mode of solid tumor cell death after γ-irradiation, a widely used treatment of cancer. Whereas the mechanisms that underlie the early γ-irradiation-induced cell death are well documented, those that drive the delayed cell death are largely unknown. Here we show that the Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-α death receptor pathways mediate the delayed cell death observed after γ-irradiation of breast cancer cells. Early after irradiation, we observe the increased expression of Fas, TRAIL-R and TNF-R that first sensitizes cells to apoptosis. Later, the increased expression of FasL, TRAIL and TNF-α permit the apoptosis engagement linked to mitotic catastrophe. Treatments with TNF-α, TRAIL or anti-Fas antibody, early after radiation exposure, induce apoptosis, whereas the neutralization of the three death receptors pathways impairs the delayed cell death. We also show for the first time that irradiated breast cancer cells excrete soluble forms of the three ligands that can induce the death of sensitive bystander cells. Overall, these results define the molecular basis of the delayed cell death of irradiated cancer cells and identify the death receptors pathways as crucial actors in apoptosis induced by targeted as well as non-targeted effects of ionizing radiation

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed.publishedVersio

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience

Mecanismes de resistance a l'adriamycine dans des cellules malignes humaines cultivees in vitro

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78611 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc