Claudia Fournier

Targeted Irradiation of Mammalian Cells Using a Heavy-Ion Microprobe

Abstract Heiß, M., Fischer, B. E., Jakob, B., Fournier, C., Becker, G. and Taucher-Scholz, G. Targeted Irradiation of Mammalian Cells Using a Heavy-Ion Microprobe. Radiat. Res. 165, 231–239 (2006). The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to ∼15,000 keV/μm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei.

Targeting head and neck cancer stem cells to overcome resistance to photon and carbon ion radiation.

Although promising new radiation therapy techniques such as hadrontherapy are currently being evaluated in the treatment of head and neck malignancies, local control of head and neck squamous cell carcinoma (HNSCC) remains low. Here, we investigated the involvement of cancer stem-like cells (CSCs) in a radioresistant HNSCC cell line (SQ20B). Stem-like cells SQ20B/SidePopulation(SP)/CD44+/ALDHhigh were more resistant to both photon and carbon ion irradiation compared with non-CSCs. This was confirmed by a BrdU labeling experiment, which suggests that CSCs were able to proliferate and to induce tumorigenicity after irradiation. SQ20B/SP/CD44+/ALDHhigh were capable of an extended G2/M arrest phase in response to photon or carbon ion irradiation compared with non-CSCs. Moreover, our data strongly suggest that resistance of CSCs may result from an imbalance between exacerbated self-renewal and proliferative capacities and the decrease in apoptotic cell death triggering. In order to modulate these processes, two targeted pharmacological strategies were tested. Firstly, UCN-01, a checkpoint kinase (Chk1) inhibitor, induced the relapse of G2/M arrest and radiosensitization of SQ20B-CSCs. Secondly, all-trans retinoic acid (ATRA) resulted in an inhibition of ALDH activity, and induction of the differentiation and radiosensitization of SQ20B/SP/CD44+/ALDHhigh cells. The combination of ATRA and UCN-01 treatments with irradiation drastically decreased the surviving fraction at 2Gy of SQ20B-CSCs from 0.85 to 0.38 after photon irradiation, and from 0.45 to 0.21 in response to carbon ions. Taken together, our results suggest that the combination of UCN-01 and ATRA represent a promising pharmacological-targeted strategy that significantly sensitizes CSCs to photon or carbon ion radiation.

Cell cycle-related bystander responses are not increased with LET after heavy-ion irradiation.

Abstract Fournier, C., Becker, D., Winter, M., Barberet, P., Heiß, M., Fischer, B., Topsch, J. and Taucher-Scholz, G. Cell Cycle-Related Bystander Responses are not Increased with LET after Heavy-Ion Irradiation. Radiat. Res. 167, 194–206 (2007). Evidence has accumulated that irradiated cells affect their unirradiated neighbors, so that they in turn display cellular responses typically associated with direct radiation exposure. These responses are generally known as bystander effects. In this study, cell cycle-related bystander responses were investigated in three strains of human fibroblasts after exposure to densely ionizing radiation. Varying the linear energy transfer (LET) from 11 to 15,000 keV μm−1 allowed a study of the impact of the complexity of DNA damage in the inducing cells on the responses of bystander cells. Using both broad-beam and microbeam irradiation, transient bystander responses were obtained for the induction of CDKN1A (p21). The latter was also observed when the transmission of bystander signals was limited to soluble factors. Targeted irradiation of single cells in confluent cell monolayers revealed no correlation between the amount of CDKN1A protein in the bystander cells and the radial distance to the targeted cells. In line with the induction of CDKN1A in bystander cells after irradiation with different LETs, a transient delay in the first G1 phase after irradiation of G0/G1 cells was observed. However, the CDKN1A induction revealed no significant effect on premature terminal differentiation considered to underlie fibrosis in irradiated tissue. Thus the unchanged differentiation pattern in bystander cells does not indicate pronounced, long-lasting effects.

Different Mechanisms of Cell Death in Radiosensitive and Radioresistant P53 Mutated Head and Neck Squamous Cell Carcinoma Cell Lines Exposed to Carbon Ions and X-Rays

PURPOSE We initiated studies on the mechanisms of cell death in head and neck squamous cell carcinoma cell lines (HNSCC) since recent clinical trials have shown that local treatment of HNSCC by carbon hadrontherapy is less efficient than it is in other radioresistant cancers. METHODS AND MATERIALS Two p53-mutated HNSCC cell lines displaying opposite radiosensitivity were used. Different types of cell death were determined after exposure to carbon ions (33.6 and 184 keV/microm) or X-rays. RESULTS Exposure to radiation with high linear energy transfer (LET) induced clonogenic cell death for SCC61 (radiosensitive) and SQ20B (radioresistant) cells, the latter systematically showing less sensitivity. Activation of an early p53-independent apoptotic process occurred in SCC61 cells after both types of irradiation, which increased with time, dose and LET. In contrast, SQ20B cells underwent G2/M arrest associated with Chk1 activation and Cdc2 phosphorylation. This inhibition was transient after X-rays, compared with a more prolonged and LET-dependent accumulation after carbon irradiation. After release, a LET-dependent increase of polyploid and multinucleated cells, both typical signs of mitotic catastrophe, was identified. However, a subpopulation of SQ20B cells was able to escape mitotic catastrophe and continue to proliferate. CONCLUSIONS High LET irradiation induced distinct types of cell death in HNSCC cell lines and showed an increased effectiveness compared with X-rays. However, the reproliferation of SQ20B may explain the potential locoregional recurrence observed among some HNSCC patients treated by hadrontherapy. An adjuvant treatment forcing the tumor cells to enter apoptosis may therefore be necessary to improve the outcome of radiotherapy.

Response of human hematopoietic stem and progenitor cells to energetic carbon ions

Purpose: To characterise the radiation response of human hematopoietic stem and progenitor cells (HSPC) with respect to X and carbon ion irradiation. Materials and methods: HSPC from peripheral blood of healthy donors treated with granulocyte-colony stimulating factor (G-CSF) were enriched for the transmembrane glycoprotein CD34 (cluster of differentiation) and irradiated with X rays or carbon ions (29 keV/μm monoenergetic beam and 60-85 keV/μm spread-out Bragg peak), mimicking radiotherapy conditions. Apoptotic cell death, cell cycle progression and the frequency of chromosomal aberrations were determined. Results: After radiation exposure no inhibition in the progression of the cell cycle was detected. However, an enhanced frequency of apoptotic cells and an increase in aberrant cells were observed, both effects being more pronounced for carbon ions than X rays, resulting in a relative biological effectiveness (RBE) of 1.4–1.7. The fraction of complex-type aberrations was higher following carbon ion exposure. Conclusions: RBE values of carbon ions are low, as expected for radiosensitive cells. The observed frequencies of apoptotic cells and chromosome aberrations in HSPC are similar to those reported for human peripheral blood lymphocytes suggesting that at least with respect to apoptosis and chromosomal aberrations mature lymphocytes reflect the respective radiation responses of their proliferating progenitors.

Atrioventricular Node Ablation in Langendorff-Perfused Porcine Hearts Using Carbon Ion Particle Therapy

Background—Particle therapy, with heavy ions such as carbon-12 (12C), delivered to arrhythmogenic locations of the heart could be a promising new means for catheter-free ablation. As a first investigation, we tested the feasibility of in vivo atrioventricular node ablation, in Langendorff-perfused porcine hearts, using a scanned 12C beam. Methods and Results—Intact hearts were explanted from 4 (30–40 kg) pigs and were perfused in a Langendorff organ bath. Computed tomgraphic scans (1 mm voxel and slice spacing) were acquired and 12C ion beam treatment planning (optimal accelerator energies, beam positions, and particle numbers) for atrioventricular node ablation was conducted. Orthogonal x-rays with matching of 4 implanted clips were used for positioning. Ten Gray treatment plans were repeatedly administered, using pencil beam scanning. After delivery, positron emission tomography-computed tomgraphic scans for detection of &bgr;+ (11C) activity were obtained. A 12C beam with a full width at half maximum of 10 mm was delivered to the atrioventricular node. Delivery of 130 Gy caused disturbance of atrioventricular conduction with transition into complete heart block after 160 Gy. Positron emission computed tomgraphy demonstrated dose delivery into the intended area. Application did not induce arrhythmias. Macroscopic inspection did not reveal damage to myocardium. Immunostaining revealed strong &ggr;H2AX signals in the target region, whereas no &ggr;H2AX signals were detected in the unirradiated control heart. Conclusions—This is the first report of the application of a 12C beam for ablation of cardiac tissue to treat arrhythmias. Catheter-free ablation using 12C beams is feasible and merits exploration in intact animal studies as an energy source for arrhythmia elimination.

Carbon ion radiotherapy of human lung cancer attenuates HIF-1 signaling and acts with considerably enhanced therapeutic efficiency

Carbon ion irradiation is an emerging therapeutic option for various tumor entities. Radiation resistance of solid tumors toward photon irradiation is caused by attenuation of DNA damage in less oxygenated tumor areas and by increased hypoxia‐inducible factor (HIF)‐1 signaling. Carbon ion irradiation acts independently of oxygen;however, the role of HIF‐1 is unclear. We analyzed the effect of HIF‐1 signaling after carbon ions in comparison to photons by using biological equivalent radiation doses in a human non‐small‐cell cancer model. The studies were performed in cultured A549 and H1299 cell lines and in A549 xenografts. Knockdown of HIF‐1α in vivo combined with photon irradiation delayed tumor growth (23 vs. 13 d; P<0.05). Photon irradiation induced HIF‐1α and target genes, predominantly in oxygenated cells (1.6‐fold; P<0.05), with subsequent enhanced tumor angiogenesis (1.7‐fold; P<0.05). These effects were not observed after carbon ion irradiation. Micro‐DNA array analysis indicated that photons, but not carbon ions, significantly induced components of the mTOR (mammalian target of rapamycin) pathway (gene set enrichment analysis; P<0.01) as relevant for HIF‐1α induction. After carbon ion irradiation in vivo, we observed substantially decreased HIF‐1α levels (8.9‐fold; P<0.01) and drastically delayed tumor growth (P<0.01), an important finding that indicates a higher relative biological effectiveness (RBE) than anticipated from the cell survival data. Taken together, the evidence showed that carbon ions mediate an improved therapeutic effectiveness without tumor‐promoting HIF‐1 signaling.—Subtil, F. S. B., Wilhelm, J., Bill, V., Westholt, N., Rudolph, S., Fischer, J., Scheel, S., Seay, U., Fournier, C., Taucher‐Scholz, G., Scholz, M., Seeger, W., Engenhart‐Cabillic, R., Rose, F., Dahm‐Daphi, J., Hänze, J. Carbon ion radiotherapy of human lung cancer attenuates HIF‐1 signaling and acts with considerably enhanced therapeutic efficiency. FASEB J. 28, 1412–1421 (2014). www.fasebj.org

The Fate of a Normal Human Cell Traversed by a Single Charged Particle

The long-term “fate” of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability.

Radiation-induced premature senescence is associated with specific cytogenetic changes.

In the present study, we set out to investigate cytogenetic changes in the progeny of two normal human fibroblast cell strains after exposure to sparsely or densely ionizing irradiation (X-rays or 9.8 MeV u(-1) carbon ions). The cells were regularly subcultured up to senescence. The transition to senescence was determined by measurement of population doubling numbers and senescence associated (SA) beta-galactosidase activity. Chromosomal changes (structural aberrations, tetraploidy) were investigated by solid staining. In temporal proximity to senescence, we observed for all populations of the two fibroblasts cell strains an increase in the fraction of cells with structural and numerical aberrations. The observed changes in the yield of structural chromosomal aberrations were similar for the progeny of controls and irradiated cells, except that a previous irradiation with a high, fractionated X-ray dose resulted in a stronger increase. Noteworthy, delayed tetraploidy in the descendants of irradiated cells exceeded the level in control cells. In addition, tetraploidy and the time of onset of senescence were significantly correlated for all populations, regardless of a preceding radiation exposure. However, the time of the onset of senescence depends on previous exposure to radiation. We conclude that the occurrence of tetraploidy is associated with senescence independently of exposure to radiation.

NF-κB-dependent DNA damage-signaling differentially regulates DNA double-strand break repair mechanisms in immature and mature human hematopoietic cells

Hematopoietic stem and progenitor cells (HSPC), that is, the cell population giving rise not only to all mature hematopoietic lineages but also the presumed target for leukemic transformation, can transmit (adverse) genetic events, such as are acquired from chemotherapy or ionizing radiation. Data on the repair of DNA double-strand-breaks (DSB) and its accuracy in HSPC are scarce, in part contradictory, and mostly obtained in murine models. We explored the activity, quality and molecular components of DSB repair in human HSPC as compared with mature peripheral blood lymphocytes (PBL). To consider chemotherapy/radiation-induced compensatory proliferation, we established cycling HSPC cultures. Comparison of pathway-specific repair activities using reporter systems revealed that HSPC were severely compromised in non-homologous end joining and homologous recombination but not microhomology-mediated end joining. We observed a more pronounced radiation-induced accumulation of nuclear 53BP1 in HSPC relative to PBL, despite evidence for comparable DSB formation from cytogenetic analysis and γH2AX signal quantification, supporting differential pathway usage. Functional screening excluded a major influence of phosphatidylinositol-3-OH-kinase (ATM/ATR/DNA-PK)- and p53-signaling as well as chromatin remodeling. We identified diminished NF-κB signaling as the molecular component underlying the observed differences between HSPC and PBL, limiting the expression of DSB repair genes and bearing the risk of an inaccurate repair.