DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

The ICRP, MELODI, and ALLIANCE workshop on effects of ionizing radiation exposure in offspring and next generations: a summary of discussions.

PURPOSE: Task Group 121 - Effects of ionizing radiation exposure in offspring and next generations - is a task group under the Committee 1 of the International Commission on Radiological Protection (ICRP), approved by the Main Commission on 18th November 2021. The main goals of Task Group 121 are to (1) review and update the scientific literature of relevance to radiation-related effects in the offspring of parent(s) exposed to ionizing radiation in both human and non-human biota; (2) to assess preconceptional and intrauterine effects of radiation exposure and related morbidity and mortality; and, (3) to provide advice about the level of evidence and how to consider these preconceptional and postconceptional effects in the system of radiological protection for humans and non-human biota. METHODS: The Task Group is reviewing relevant literature since Publication 90 'Biological effects after prenatal irradiation (embryo and fetus)' (2003) and will include radiation-related effects on future generations in humans, animals, and plants. This review will be conducted to account for the health effects on offspring and subsequent generations in the current system of radiological protection. Radiation detriment calculation will also be reviewed. Finally, preliminary recommendations will be made to update the integration of health effects in offspring and next generations in the system of radiological protection. RESULTS: A Workshop, jointly organized by ICRP Task Group 121 and European Radiation Protection Research Platforms MELODI and ALLIANCE was held in Budapest, Hungary, from 31st May to 2nd June 2022. Participants discussed four important topics: (1) hereditary and epigenetic effects due to exposure of the germ cell line (preconceptional exposure), (2) effects arising from exposure of the embryo and fetus (intrauterine exposure), (3) transgenerational effects on biota, and (4) its potential impact on the system of radiological protection. CONCLUSIONS: Based on the discussions and presentations during the breakout sessions, newer publications, and gaps on the current scientific literature were identified. For instance, there are some ongoing systematic reviews and radiation epidemiology reviews of intrauterine effects. There are newer methods of Monte Carlo simulation for fetal dosimetry, and advances in radiation genetics, epigenetics, and radiobiology studies. While the current impact of hereditary effects on the global detriment was reported as small, the questions surrounding the effects of radiation exposure on offspring and the next generation are crucial, recurring, and with a major focus on exposed populations. This article summarizes the workshop discussions, presentations, and conclusions of each topic and introduces the special issue of the International Journal of Radiation Biology resulting from the discussions of the meeting.

Cryopreservation of neurospheres derived from human glioblastoma multiforme.

Cancer stem cells have been shown to initiate and sustain tumor growth. In many instances, clinical material is limited, compounded by a lack of methods to preserve such cells at convenient time points. Although brain tumor-initiating cells grown in a spheroid manner have been shown to maintain their integrity through serial transplantation in immune-compromised animals, practically, it is not always possible to have access to animals of suitable ages to continuously maintain these cells. We therefore explored vitrification as a cryopreservation technique for brain tumor-initiating cells. Tumor neurospheres were derived from five patients with glioblastoma multiforme (GBM). Cryopreservation in 90% serum and 10% dimethyl sulfoxide yielded greatest viability and could be explored in future studies. Vitrification yielded cells that maintained self-renewal and multipotentiality properties. Karyotypic analyses confirmed the presence of GBM hallmarks. Upon implantation into NOD/SCID mice, our vitrified cells reformed glioma masses that could be serially transplanted. Transcriptome analysis showed that the vitrified and nonvitrified samples in either the stem-like or differentiated states clustered together, providing evidence that vitrification does not change the genotype of frozen cells. Upon induction of differentiation, the transcriptomes of vitrified cells associated with the original primary tumors, indicating that tumor stem-like cells are a genetically distinct population from the differentiated mass, underscoring the importance of working with the relevant tumor-initiating population. Our results demonstrate that vitrification of brain tumor-initiating cells preserves the biological phenotype and genetic profiles of the cells. This should facilitate the establishment of a repository of tumor-initiating cells for subsequent experimental designs.

hTERT overexpression alleviates intracellular ROS production, improves mitochondrial function, and inhibits ROS-mediated apoptosis in cancer cells.

The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase holoenzyme. Evidence is accumulating to link hTERT to activities other than telomere maintenance and immortalization. Here, we show that hTERT overexpression not only reduces the basal cellular reactive oxygen species (ROS) levels but also inhibits endogenous ROS production in response to stimuli that induce intracellular ROS generation. Conversely, siRNA-mediated gene silencing of hTERT potentiated the increase in cellular ROS levels following exposure to oxidative stress. This antioxidant effect of hTERT is mediated via a significant increase in the ratio of reduced to oxidized glutathione (GSH:GSSG) as well as efficient recovery of the oxidized peroxiredoxin to its nonoxidized form. Our data also provide evidence for mitochondrial localization of hTERT, and a significantly higher activity of cytochrome C oxidase, the rate-limiting enzyme in the mitochondrial electron transport chain, in hTERT overexpressing cells. To ascertain whether the improved mitochondrial function and antioxidant effect of hTERT could provide cancer cells with a survival advantage, the effect of oxidative stress on mitochondrial apoptosis was evaluated. Indeed, hTERT overexpressing cells inhibited cytosolic acidification, translocation of Bax, the drop in mitochondrial transmembrane potential, the release of cytochrome C to the cytosol, and cell death. Taken together, these data demonstrate a hitherto undefined role of hTERT in alleviating cellular ROS levels by way of potentiating the cellular antioxidant defense systems, and in doing so endowing cancer cells with the ability to evade death stimuli.

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.