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Purpose: The efficacy of DNA-targeting radionuclide therapies might be strongly enhanced by employing short range particle-emitters. However, the gain of effectiveness is not yet well substantiated. We compared the Auger electron emitter I-125 to the ß - -emitter P-32 in terms of biological effectiveness per decay and radiation dose when located in the close proximity to DNA using DNA Triplex-forming oligonucleotides (TFO). The clonogenicity and the induction of DNA double-strand breaks (DSB) were investigated in SCL-II cells after exposure to P-32- or I-125-labeled TFO targeting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and after external homogeneous exposure to gamma-rays as reference radiation., Materials and Methods: TFO were labeled with P-32 or I-125 using the primer extension method. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in SCL-II cells., Results: I-125-TFO induced a pronounced decrease of cell survival (D 37 at ∼360 accumulated decays per cell, equivalent to 1.22 Gy cell nucleus dose) and a significant increase of 53BP1 foci with increasing decays. The P-32-labeled TFO induced neither a strong decrease of cell survival nor an increase of 53BP1 foci up to ∼4000 accumulated decays per cell, equivalent to ∼1 Gy cell nucleus dose. The RBE for I-125-TFO was in the range of 3-4 for both biological endpoints., Conclusions: I-125-TFO proved to be much more radiotoxic than P-32-TFO per decay and per unit dose although targeting the same sequence in the GAPDH gene. This might be well explained by the high number of low energy Auger electrons emitted by I-125 per decay, leading to a high ionization density in the immediate vicinity of the decay site, probably producing highly complex DNA lesions overcharging DNA repair mechanisms.

BCL-2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL-2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL-2 by its competitive ligand HA14-1. Inhibition of BCL-2 caused a diminished BCL-2 expression and higher levels of cleaved BAX, activated Caspase-3 and cleaved PARP, all pro-apoptotic markers, when compared with untreated differentiating cells. In parallel, flow cytometric analysis of HA14-1-treated cells revealed a delayed differentiation into HuC/D+ neuronal cells when compared to untreated differentiating cells. In conclusion, BCL-2 possess a protective function in fully differentiated ReNcell VM cells. We propose that the pro-survival signaling of BCL-2 is closely connected with its stimulatory effects on neurogenesis of human neural progenitor cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis. Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10 μM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis. Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells. Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription., (Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.)

Objective: To study the biological effectiveness of Auger electrons emitted by (99m)Tc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation., Material and Methods: Cells were either gamma(60Co)-irradiated (0.67 Gy/min) or exposed to (99m)Tc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of (99m)Tc under the given geometry., Results: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SF 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The (99m)Tc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure., Conclusion: No overall increased biological effectiveness due to the emitted Auger electrons of (99m)Tc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after (99m)Tc-exposure at very low doses has to be further investigated.

Cell survival, induction of apoptosis, and micronucleus formation have been examined in non-transformed human amnion fluid fibroblast-like (AFFL) cells and in a human squameous cell carcinoma (SCL-II) cell line after exposure to the Auger electron emitter (65)Zn and after external low-LET radiation. Cellular uptake and subcellular distribution of (65)Zn(2+) were studied in vitro and the absorbed radiation dose was calculated applying analytical dosimetry models. Auger electrons generated during decay of (65)Zn induced a prominent decrease in cell survival and increased the levels of apoptotic as well as micronucleated cells when compared to external low-LET irradiation. Relative biological effectiveness has been determined for cell survival (RBE approximately 4), micronucleus formation (RBE approximately 2) and apoptosis induction (RBE approximately 5-8) in SCL-II cells and for micronucleus formation (RBE approximately 4-5) and apoptosis induction (RBE approximately 6-10) in AFFL cells, respectively. This demonstrates a general enhanced biological effectiveness of (65)Zn in both investigated cell lines when compared to external low-LET radiation. The distribution pattern of intracellular Zn(2+) was found to be non-uniform, showing enhanced amounts of Zn(2+) in the perinuclear region and low amounts inside the cell nucleus, suggesting a major energy deposition close to the nuclear envelope.

Induction of apoptosis and micronucleus formation has been studied in a transformed human squamous cell carcinoma cell line (SCL-II) after exposure to the Auger electron emitter Zinc-65 (65Zn) and after external low-LET radiation. Exposure to non-radioactive Zn and unirradiated cells served as controls. Studies on the cellular uptake of 65Zn2+ have been carried out in vitro and conventional dosimetric models have been applied to derive the absorbed radiation dose. Auger electrons, generated during decay of 65Zn2+, are strong inducers of micronuclei as well as of apoptosis, in comparison to external low-LET irradiation. The relative biological effectiveness has been determined and was found to be in the range of 1.2-2.7 for the two investigated biological endpoints, depending on which mathematical model for describing the dose-effect curves was used. A non-uniform distribution of intracellular Zn2 + was observed, showing a strong signal in the perinuclear region. We conclude that separate radiation weighting factors for Auger electrons should be established depending on the nuclide and its ability to interact with the DNA (e.g. 65Zn by Zinc-finger proteins).

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1-1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects.

Objective: To study the biological effectiveness of Auger electrons emitted by 99mTc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation. Material and methods: Cells were either gamma(60Co)-irradiated 0.67 Gy/min) or exposed to 99mTc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of 99mTc under the given geometry. Results: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SP = 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The 99mTc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure. Conclusion: No overall increased biological effectiveness due to the emitted Auger electrons of 99mTc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after 99mTc-exposure at very low doses has to be further investigated. [ABSTRACT FROM AUTHOR]