DosiKit, a New Immunoassay for Fast Radiation Biodosimetry of Hair and Blood Samples.

DosiKit is a field radiation biodosimetry immunoassay for fast triage of individuals exposed to external total-body or partial-body irradiation (TBI or PBI). Assay proof-of-concept based on γ-H2AX analysis of human blood samples has been previously described as a promising tool for rapid assessment of TBI. Here, we report on the performance of the assay for PBI based on an analysis of hair follicles irradiated with a 137Cs gamma-ray source, at doses ranging from 0 to 20 Gy and dose rates ranging from ∼0.8 to ∼3 Gy/min. First, we show that the DosiKit protocol allows extraction and analysis of hair follicle proteins. Next, we show that irradiated hair follicles trigger a DNA damage response by inducing dose-dependent γ-H2AX expression. Since γ-H2AX expression strongly decreases 2 to 4 h postirradiation, due to DNA repair, we hypothesized that an antibody targeting the S*/T*Q domains, phosphorylated by ATM for DNA repair activation (pSQTQ), would extend the postirradiation dosimetry time window. DosiKit analysis of pSQTQ in ex vivo irradiated cynomolgus monkey skin explants shows that these sequences are phosphorylated in a dose-dependent manner up to 8 h postirradiation, and that statistically different ranges of external radiation exposure can be distinguished (0-2 Gy, 5-10 Gy, 20 Gy). Since the DosiKit protocol is intended to be used on both blood and hair samples, we also show that SQTQ sequences are phosphorylated dose-dependently in human blood, allowing samples to be classified into three radiation dose ranges (0-0.1 Gy, 0.5-3 Gy and 5-8 Gy). In conclusion, radiation biodosimetry can be performed on both blood and hair samples up to 8 h after exposure using the DosiKit protocol, allowing the concomitant characterization of TBI and PBI for fast and efficient radiological crisis management.

Breast cancer stem cell-like cells generated during TGFß-induced EMT are radioresistant.

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24-/low/CD44+ CSCs and their corresponding CD24+/CD44low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor ß (TGFß) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFß-induced cell reprogramming. We showed that mesenchymal CD24-/low/CD44+ CSCs are more resistant to radiation compared with CD24+/CD44low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24-/low/CD44+ cells acquired during EMT.

DosiKit, a New Portable Immunoassay for Fast External Irradiation Biodosimetry.

DosiKit is a new field-radiation biodosimetry immunoassay for rapid triage of individuals exposed to external total-body irradiation. Here, we report on the validation of this immunoassay in human blood cell extracts 0.5 h after in vitro exposure to 137Cs gamma rays, using γ-H2AX analysis. First, calibration curves were established for five donors at doses ranging from 0 to 10 Gy and dose rates ranging from ∼0.8 to ∼3 Gy/min. The calibration curves, together with a γ-H2AX peptide scale, enabled the definition of inter-experimental correction factors. Using previously calculated correction factors, blind dose estimations were performed at 0.5 h postirradiation, and DosiKit performance was compared against concomitant dicentric chromosome assay (DCA), the current gold standard for external irradiation biodosimetry. A prototype was then assembled and field tested. We show that, despite significant inter-individual variations, DosiKit can estimate total-body irradiation doses from 0.5 to 10 Gy with a strong linear dose-dependent signal and can be used to classify potentially exposed individuals into three dose ranges: below 2 Gy, between 2 and 5 Gy and above 5 Gy. The entire protocol can be performed in 45 min, from sampling to dose estimation, with a new patient triaged every 10 min. While DCA enables precise measurement of doses below 5 Gy, it is a long and difficult method. In contrast, DosiKit is a quick test that can be performed directly in the field by operational staff with minimal training, and is relevant for early field triage and identification of individuals most likely to experience acute radiation syndrome. These findings suggest that DosiKit and DCA are complementary and should be combined for triage in a mass scale event. While the proof-of-concept reported here validates the use of DosiKit at 0.5 h postirradiation, further studies are needed to calibrate and evaluate the performance of the DosiKit assay at longer times after irradiation.

Forced extinction of CD24 stem-like breast cancer marker alone promotes radiation resistance through the control of oxidative stress.

Along with CD44, CD24 is a key marker of breast cancer stem cells (CSCs), frequently defined by CD24(-)/CD44(+) labeling. Among all phenotypes classically attributed to breast CD24(-)/CD44(+) cancer cells, radiation resistance has been extensively described and seen as being implicated in radiotherapy failure. Our previous data indicated that CD24(-) cells constitute a radiation-resistant subpopulation transitory selected by high doses of ionizing radiation. However, little is known about the biological role of CD24 in breast cancers, and no function has been assigned to CD24 in radiation response. Here, CD24 expression was induced in CD24(-) cells or knocked-down in CD24(+) cells. We show that forced extinction of CD24 expression is associated with decreased proliferation rate, lower levels of reactive oxygen species (ROS) and decreased genomic instability. On the opposite when CD24 is artificially expressed in CD24(-) cells, proliferation rates in vitro and in vivo, ROS levels and genomic instability are enhanced. Moreover, we observe that loss of CD24 expression leads to radiation resistance, by preventing radiation-induced cell death and promoting generation of progeny in relation to lower G2/M blockade and a smaller proportion of polyploid cells. Finally, control of ROS levels appears to be the key event in the CD24-mediated radiation response. For the first time, CD24 is proposed as a direct actor in radiation response of breast cancer cells, independently of CD44 expression. These findings could have interesting applications in evaluating the intrinsic radiation response of primary tumors.

A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

In vitro aging of rat lung cells. Downregulation of telomerase activity and continuous decrease of telomere length are not incompatible with malignant transformation.

Most normal mammalian somatic cells cultivated in vitro enter replicative senescence after a finite number of divisions, as a consequence of the progressive shortening of telomeres during proliferation that reflects one aspect of organism/cellular aging. The situation appears more complex in rodent cells due to physiological telomerase expression in most somatic normal tissues, great telomere length, and the difficulties of finding suitable in vitro culture conditions. To study in vitro aging of rat lung epithelial cells, we have developed primary culture conditions adapted to rat fresh lung explants and have studied for 1 year (50 passages) the changes in cellular proliferation and mortality, genetic instability, telomerase activity, telomere length, and tumorigenic potential. We have observed an absence of senescence and/or crisis, a transient genetic instability, the persistence of a differentiated Clara cell phenotype, a steady decrease in telomerase activity followed by a low residual activity together with a continuous decrease in telomere length, a constant rate of proliferation, and the acquisition of tumorigenic potential. The bypass of the growth arrest and the acquisition of long-term growth properties could be explained by the loss of p16(INK4a) expression, the ARF/p53 pathway not being altered. In conclusion, these results clearly indicate that, in rat lung epithelial cells, in vitro transformation and acquisition of tumorigenic properties can occur even if the telomere length is still decreasing and telomerase activity remains downregulated.