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The Auger effect is not yet fully understood in respect to the deposited energy as well as to the dose rate. Therefore, we studied the Auger electron emitter (AEE) I-123 and I-125 which are characterized by a different half-life (13.2 h vs. 59.4 d) and by different average numbers of Auger electrons emitted per decay (ratio I-123/I-125 ~ 1:2). The biological response in mammalian cells labelled with various activity concentrations of 5-(123)iodine-2'-deoxyuridine (I-123-UdR) and (5-(125)iodine-2'-deoxyuridine (I-125-UdR) was thoroughly investigated to further elucidate the biological effectiveness of these particular electron emitters. SCL-II cells were synchronized in G1-cell cycle phase, subsequently labelled with I-123- respectively I-125-UdR and the cellular uptake and DNA incorporation of I-UdR was determined. Chromatin damage was quantified by the alkaline Comet assay, apoptosis induction assessed by the Annexin V/PI assay employing flow cytometry and micronucleus formation was quantified using the Cytochalasin-B micronucleus assay at various times post-labelling. Cs-137 γ-rays served as reference radiation.I-125-UdR caused pronounced apoptosis when compared to !-123-UdR. Micronucleus induction and chromatin damage was very similar for both radionuclides. Both AEE caused a pronounced and long-lasting G2/M cell cycle arrest. On average one decay of I-125 every 120 seconds in the DNA of a single cell is sufficient to induce a permanent G2/M cell cycle arrest in SCL-II cells.Albeit of a lower dose rate, I-125-UdR is more cytotoxic in comparison with I-123-UdR.Funded by Bundesministerium für Umwelt, Natur und Reaktorsicherheit (BMU), Bundesamt für Strahlenschutz; Project No.: 3608S03002

Introduction: Non-targeted effects caused by signalling of directly irradiated cells to neighboring non-irradiated cells, termed “bystander effect”, is still controversially discussed, especially in radiotherapy. Xue et al. (2002) demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated I-125 inhibits growth of co-injected LS174T cells (inhibitory bystander effect). Kishikawa et al. (2006) from the same group demonstrated in the same experimental set-up that I-123, an Auger electron emitter (AEE) whose emission spectrum is similar to that of I-125, stimulates the proliferation of neighboring unlabeled tumor cells (stimulatory bystander effect) and attributed the effect to soluble factors. To elucidate the nature of the opposed bystander effect for I-123 and I-125, we studied the cell culture medium-mediated bystander effect in respect to micronucleus formation and apoptosis in two mammalian cell lines.Methods: Medium from AEE-treated human SCL-II and Kidney-T1 cells (I-123-UdR: 16, 48, 80 kBq/ml; I-125-UdR: 8, 24, 40 kBq/ml) was harvested and transferred to the respective non-irradiated bystander cell cultures. Micronucleus formation and apoptosis induction was assessed by fluorescence microscopy (Hoechst-33342 DNA staining) respectively flow cytometry (Annexin V/PI assay) for up to 48 h.Results: No significant bystander effect in respect to micronucleus formation and apoptosis induction in neither cell-line was observed.Conclusions: Neither I-123 nor I-125 have been found to induce cell culture medium-mediated bystander effects in the two investigated cell lines. However, in consideration of publications with contrary findings, the specific cell line respectively culture or experimental conditions may be of importance for inducing bystander effects. Other than stated the reported findings by Xue et al. (2002) and Kishikawa et al. (2006) might also be due to cell-cell contact mediated bystander effects. Funded by Bundesamt für Strahlenschutz; Bundesministerium für Umwelt, Natur und Reaktorsicherheit (BMU); Project No.: 3608S03002Referenzen:Xue et al. Proc. Natl. Acad. Sci. USA 99, 13765-13770, 2002Kishikawa et al. Radiat. Res.;165(6):688-94, 2006

Introduction: Theoretical calculations and experimental findings suggest that DNA-incorporated Auger electron emitter (AEE) cause primarily complex DNA lesions. Recent cell cycle analysis revealed, that 5-(125)iodine-2'-deoxyuridine (I-125-UdR)-exposed SCL-II cells display a pronounced and rather long-lasting G2/M cell cycle arrest. We studied therefore in more detail whether the observed G2/M arrest is of permanent or temporary nature and thereby examined the average number of decay per cell necessary to induce a long-lasting cell cycle arrest in SCL-II cells.Methods: SCL-II cells were synchronized in G1-phase and incubated with 0.1, 1, 4 and 8 kBq/ml of I-125-UdR during S-phase so that approximately 90 % of the cells were subsequently labeled. Cell cycle analysis was performed by flow cytometry (Click-iT EdU cell proliferation assay and Sytox Green nucleic acid stain) up to 4 d post-labeling with a constant sampling every 8 h post-labeling.Results: DNA-incorporated I-125-UdR decelerated SCL-II cell cycle progression. Cell cycle perturbances were observed and a pronounced and prolonged G2/M-phase arrest was detected at activity concentration of 4 kBq/ml I-125-UdR. Up to 70 % of the cells were arrested in G2/M-phase up to 38 h post-labeling with 8 kBq/ml I-125-UdR. Moreover, a distinct population of G2/M-arrested cells was detectable up to 94 h post-labeling. As shown by parallel EdU staining these cells were permanently caught in the first G2/M cell cycle phase post-labeling. On average one decay every 90 s per DNA/cell was calculated to induce a permanent G2/M arrest in SCL-II cells.Conclusions: Incorporated I-125-UdR induces major cell cycle perturbances in SCL-II cells. The observed permanent G2/M arrest suggests a threshold in terms of decays per time per cell and hence persisting DNA damage beyond no G2/M release seems to occur. This implies different levels of DNA damage for the induction and the release of the G2/M arrest in SCL-II cells. Funded by Bundesministerium für Umwelt, Natur und Reaktorsicherheit (BMU), Bundesamt für Strahlenschutz; Project No.: 3608S03002

Die humane neurale VM197-Progenitorzelllinie wurde bezüglich der Interkonnektivität von Wnt-gesteuerter neuraler Zelldeterminierung und Apoptose untersucht. Die Zelldifferenzierung ging mit der Aktivierung von pro- und anti-apoptotischen Proteinen und der Zunahme apoptotischen Zellverlustes einher. Differenzierte Zellen wiesen eine geringere Apoptosesuszeptibilität gegenüber proliferierenden Zellen auf. Beta-Catenin-Spaltung und eine geringere Apoptoserate nach GSK-3ß-Inhibierung in differenzierenden Zellen deuten auf eine Interkonnektivität von kanonischem Wnt-Signalweg und Apoptose hin.