Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro.

Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro.NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs.Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage.Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer.

Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles.

Differential gene expression in human fibroblasts after alpha-particle emitter (211)At compared with (60)Co irradiation.

PURPOSE: The aim of this study was to identify gene expression profiles distinguishing alpha-particle (211)At and (60)Co irradiation. MATERIALS AND METHODS: Gene expression microarray profiling was performed using total RNA from confluent human fibroblasts 5 hours after exposure to (211)At labeled trastuzumab monoclonal antibody (0.25, 0.5, and 1 Gy) and (60)Co (1, 2, and 3 Gy). RESULTS: We report gene expression profiles that distinguish the effect different radiation qualities and absorbed doses have on cellular functions in human fibroblasts. In addition, we identified commonly expressed transcripts between (211)At and (60)Co irradiation. A greater number of transcripts were modulated by (211)At than (60)Co irradiation. In addition, down-regulation was more prevalent than up-regulation following (211)At irradiation. Several biological processes were enriched for both irradiation qualities such as transcription, cell cycle regulation, and cell cycle arrest, whereas mitosis, spindle assembly checkpoint, and apoptotic chromosome condensation were uniquely enriched for alpha particle irradiation. CONCLUSIONS: LET-dependent transcriptional modulations were observed in human fibroblasts 5 hours after irradiation exposure. These findings suggest that in comparison with (60)Co, (211)At has the clearest influence on both tumor protein p53-activated and repressed genes, which impose a greater overall burden to the cell following alpha particle irradiation.

Biological consequences of formation and repair of complex DNA damage.

Endogenous processes or genotoxic agents can induce many types of single DNA damage (single-strand breaks, oxidized bases and abasic sites). In addition, ionizing radiation induces complex lesions such as double-strand breaks and clustered damage. To preserve the genomic stability and prevent carcinogenesis, distinct repair pathways have evolved. Despite this, complex DNA damage can cause severe problems and is believed to contribute to the biological consequences observed in cells exposed to genotoxic stress. In this review, the current knowledge of formation and repair of complex DNA damage is summarized and the risks and biological consequences associated with their repair are discussed.

RBE of α-particles from (211)At for complex DNA damage and cell survival in relation to cell cycle position.

PURPOSE: To investigate cell cycle effects and relative biological effectiveness (RBE) of α-particles from the clinically relevant radionuclide Astatine-211 ((211)At), using X-rays as reference radiation. Double-strand breaks (DSB), non-DSB clusters containing oxidised purines and clonogenic survival were investigated. MATERIALS AND METHODS: Asynchronous V79-379A fibroblasts or cells synchronised with mimosine in G1, early, mid and late S phase or in mitosis were irradiated with X-rays (100 kV(p)) or (211)At (mean linear energy transfer (LET) 110 keV/µm). Induction of DSB and clusters was determined using pulsed-field gel electrophoresis with fragment analysis. Cell survival was obtained with the clonogenic assay. RESULTS: In asynchronous cells RBE for DSB- and cluster-induction was 3.5 and 0.59, respectively. RBE for 37% cell survival was 8.6. In different cell cycle phases RBE varied from 1.8-3.9 for DSB and 3.1-7.9 for 37% survival (survival at 2 Gy was 6.9-38 times lower after α-irradiation). (211)At induced 6 times more DSB and X-rays induced 11 times more DSB in mitotic cells with highly compacted chromatin relative G1. CONCLUSIONS: The radio-response is cell cycle dependent and differs between proliferating and non-cycling cells for both low- and high-LET radiation, resulting in a variation in RBE of α-particles between 1.8 and 8.6.

Clustered DNA damage in irradiated human diploid fibroblasts: influence of chromatin organization.

Clustered DNA damages are induced by ionizing radiation and are defined as two or more lesions within one or two helical turns. The aim of this study was to investigate the induction and repair of clustered DNA damage in cells with emphasis on the influence of structural differences in the chromatin organization. Human fibroblasts were irradiated with X rays and induced DSBs and clustered damages were quantified using pulsed-field gel electrophoresis combined with postirradiation incubation with the base excision repair endonuclease Fpg, which recognizes oxidized purines and cleaves the strand at sites inducing strand breaks. Hence clustered damages appear in enzyme-treated samples as additional DSBs. The chromatin was modified by different pretreatments that resulted in structures with varying compactness and levels of free radical scavenging capacity. We found that the induction of DSBs and clustered damages increased linearly with dose in all structures and that both types of lesions were allocated randomly within the nucleus. The induction yields increased with decreasing compactness of chromatin, and the chromatin effect was larger for clustered lesions than for DSBs. Clustered damages were processed efficiently with a fast and a slow repair component similar to that for induced DSBs.

Influence of chromatin structure on induction of double-strand breaks in mammalian cells irradiated with DNA-incorporated 125I.

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts.

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

Chromatin organization contributes to non-randomly distributed double-strand breaks after exposure to high-LET radiation.

The influence of higher-order chromatin structure on the non-random distribution of DNA double-strand breaks induced by high-LET radiation was investigated. Five different chromatin structures (intact cells, condensed and decondensed chromatin, nucleoids and naked genomic DNA) from GM5758 cells or K562 cells were irradiated with (137)Cs gamma-ray photons and 125 keV/microm nitrogen ions (16-25 MeV/nucleon). DNA was purified with a modified lysis procedure to avoid release of heat-labile sites, and fragment size distributions and double-strand break yields were analyzed by different pulsed-field gel electrophoresis protocols. Whereas double-strand breaks in photon-irradiated cells were randomly distributed, irradiation of intact K562 cells with high-LET nitrogen ions produced an excess of non-randomly distributed DNA fragments 10 kb-1 Mbp in size. Complete removal of proteins eliminated this non-random component. There was a gradual increase in the yield of double-strand breaks for each chromatin decondensation step, and compared to intact cells, the yields for naked DNA (in buffer without scavengers) increased 83 and 25 times after photon and nitrogen-ion irradiation, respectively. The corresponding relative biological effectiveness decreased from 1.6-1.8 for intact cells to 0.49 for the naked DNA. We conclude that the organization of DNA into chromatin fiber and higher-order structures is responsible for the majority of non-randomly distributed double-strand breaks induced by high-LET radiation. However, our data suggest a complex interaction between track structure and chromatin organization over several levels.

Cleavage of cellular DNA by calicheamicin gamma1.

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0 degrees C as compared to 37 degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calicheamicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.