Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction

Purpose: Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. Experimental Design: Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. Results: Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses = 1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 x 0.25 Gy. Conclusions: We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation. (C) 2018 AACR

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway

Bioengineering strategies for regeneration of craniofacial bone: a review of emerging technologies

Oral Diseases (2010) 16 , 709–716Although advances in surgical techniques and bone grafting have significantly improved the functional and cosmetic restoration of craniofacial structures lost because of trauma or disease, there are still significant limitations in our ability to regenerate these tissues. The regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science, and engineering technology. Tissue engineering is an interdisciplinary field of study that addresses this challenge by applying the principles of engineering to biology and medicine toward the development of biological substitutes that restore, maintain, and improve normal function. This review will explore the impact of biomaterials design, stem cell biology and gene therapy on craniofacial tissue engineering.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79049/1/j.1601-0825.2010.01682.x.pd

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Aufbau und Inbetriebnahme eines kontinuierlich gepumpten Farbstofflasers

Das Institut für Festkörperforschung (IFF) der KernforschungsanlageJülich GmbH befaßt sich mit der spektroskopischenUntersuchung von Halbleitern und Halbleitergrenzflächen.Um bei diesen Untersuchungen gezielt elektronische Resonanzenabfragen zu können, sind durchstimmbare Wellenlängenim Bereich von 400nm - 1000nm erforderlich.Diese Anforderung erfüllen kontinuierlich gepumpte Farbstofflaser;da käufliche Farbstofflaser im allgemeinenkompakt gebaut sind, ist ihre Handhabung sehr schwierig.Insbesondere erweist sich die Umjustierung des Resonators,wie sie bei einem Wechsel der Betriebsart vom visuellen inden UV- bzw. IR-Spektralbereich erfolgen muß, als sehraufwendig. Da im speziellen die vertikale Geometrie derResonator-Cavity des vorhandenen kommerziellen Farbstofflasersnicht optimal ist, sollte im Rahmen dieser Diplomarbeitin Zusammenarbeit mit der Entwicklungsabteilung desInstitutes Zentralabteilung Allgemeine TechnologieMechanische Werkstätten (ZAT-MW/E) die Konstruktion einesin seiner Handhabung möglichst einfachen, gut justierbarenFarbstofflasers erfolgen.Die Messungen zur Inbetriebnahme und zur Charakterisierungder Leistungsfähigkeit erfolgten im Labor für Laserspektroskopiedes IFF

Flowcytometrical analysis of radiation-induced γ-H2AX foci

PURPOSE: Phosphorylation of histone H2AX (γ-H2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell (γ-H2AX-assay). We investigated whether the mean intensity and the mean number of radiation-induced γ-H2AX foci vary as a function of radiation quality and dose.MATERIALS AND METHODS: Jurkat cells were irradiated with different doses of either low linear energy transfer (LET) 137Cs γ-rays or high LET 241Am α-particles. The γ-H2AX foci were detected using immunocytochemistry (primary antibody Anti-phospho-Histone H2AX(Ser139) mouse IgG, secondary antibody FITC goat anti-mouse IgG) and quantified by counting the number of γ-H2AX foci employing fluorescence microscopy and by measuring the mean signal intensity in single cell nuclei by flow cytometry. RESULTS: The mean number of γ-H2AX foci is increased in a dose dependent manner for both radiation qualities and are broadly similar at identical absorbed radiation dose for both investigated radiation qualities. The mean γ-H2AX signal intensity of single nuclei is increased after alpha-irradiation when compared to γ-irradiation at the same absorbed radiation dose. CONCLUSIONS: α-particle induced γ-H2AX foci show higher signal intensities compared to γ-ray-induced γ-H2AX foci. The mean intensity of radiation-induced γ-H2AX foci is dependent on radiation quality in Jurkat cells

Quantification of γ-H2AX foci following γ-rays and α-particls in Jurkat cells

PURPOSE: Phosphorylation of histone H2AX occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We investigated whether the mean intensity measured by flow cytometry and the mean number of radiation-induced γ-H2AX foci vary as a function of radiation quality and dose. Furthermore we investigated the relation between the induction of apoptosis and the mean intensity and mean number of radiation-induced γ-H2AX foci.MATERIALS AND METHODS: Jurkat cells were irradiated with different doses of either low linear energy transfer (LET) 137Cs γ-rays or high LET 241Am α-particles. The γ-H2AX foci were detected using immunocytochemistry and quantified by measuring the mean intensity by flow cytometry and counting the number of γ-H2AX foci with a fluorescence microscope. Apoptosis 24h after irradiation was detected determining the relative DNA fragmentation rates indicative for apoptosis.RESULTS: The mean number of γ-H2AX foci increased dose dependent for both radiation qualities, but the mean intensity of γ-H2AX foci after α-radiation is much higher than after γ-radiation refered to the same dose. These data are confirmed by microscopic observations. Furthermore it seems to be that α-particles induce more apoptosis than γ-rays at the same dose and at a similar mean number of radiation-induced γ-H2AX foci.CONCLUSIONS: γ-rays and α-particles induced the phosphorylation of H2AX; the variation in the mean intensity and the mean number of radiation-induced γ-H2AX foci is dependent on radiation quality

Quantification of γ-H2AX foci after exposure to I-123-iododeoxyuridine in comparison to γ- and α-irradiation

Introduction: Phosphorylation of histone H2AX occurs at sites flanking DNA double-strand breaks (DSBs) and can provide an indirect measure for the number of DSBs within a cell. Recent publications suggest LET-dependent differences in the intensity and size of γ-H2AX foci. To determine whether γ-H2AX foci caused by DNA-associated Auger electron emitters (AEE) induce high-LET type γ-H2AX foci we investigated the mean intensity as well as the mean number of γ-H2AX foci after exposure to I-123, high- and low-LET radiation.Methods: Human T-lymphoma Jurkat cells were either exposed to I-123-iododeoxyuridine (I-123-UdR; 2-200 kBq per 10E6 cells) for 20 h or irradiated with different doses of low-LET Cs-137 γ-rays respectively high-LET Am-241 α-particles. The γ-H2AX foci were quantified by measuring the mean signal intensity using flow cytometry and by counting the number of γ-H2AX foci microscopically by eye. Co-localization experiments were performed with the DNA-repair associated protein 53BP1 employing confocal microscopy.Results: The mean numbers of γ-H2AX foci per cell showed a much more pronounced increase after exposure to I-123 when compared to γ- and α-irradiation. However, the mean intensity of γ H2AX signals per cell nucleus, was very similar after I-123 and α-particle exposure. The individual γ H2AX foci induced by I-123 resemble γ H2AX foci induced by γ-rays and appear to be smaller, more distinct and/or less intense stained than those after α-irradiation. 53BP1 foci do not always co-localize with γ-H2AX foci. Conclusions: The presumed complexity of the DNA-lesion caused by DNA-associated AEE is not reflected in the size and the intensity of γ-H2AX foci.Funded by Bundesministerium für Bildung und Forschung (BMBF), Project No.: 02NUK005

Quantification of γ-H2AX foci following γ-rays and α-particles in Jurkat cells

PURPOSE: Phosphorylation of histone H2AX occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We investigated whether the mean intensity measured by flow cytometry and the mean number of radiation-induced γ-H2AX foci vary as a function of radiation quality and dose. Furthermore we investigated the relation between the induction of apoptosis and radiation-induced γ-H2AX foci.MATERIALS AND METHODS: Jurkat cells were irradiated with different doses of either low linear energy transfer (LET) 137Cs γ-rays or high LET 241Am α-particles. The γ-H2AX foci were detected using immunocytochemistry and quantified by measuring the mean intensity by flow cytometry and counting the number of γ-H2AX foci with a fluorescence microscope. Apoptosis 24h after irradiation was detected via Annexin-V-FITC/ PI-assay.RESULTS: For both radiation qualities, the mean number of γ-H2AX foci is increased as a function of dose and was fairly similar at identical absorbed radiation dose. Apoptosis in Jurkat cells is more efficiently induced by α-particles at similar mean numbers of γ-H2AX foci per cell. The mean γ-H2AX signal intensity of single nuclei is increased after exposure to α-particles when compared to γ-irradiation at the same absorbed radiation dose.CONCLUSIONS: The mean intensity of radiation-induced γ-H2AX foci is dependent on radiation quality in Jurkat cells