Genomic instability after targeted irradiation of human lymphocytes: Evidence for inter-individual differences under bystander conditions

Environmental (222)radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET alpha-particle irradiation initiate deleterious genetic consequences in both radiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single He-3(2+) particle traversal to a single cell, are sufficient to Induce RIGI Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (Rid, measured as delayed chromosome aberrations) Although this was not highly significant, it was possibly masked by high levels of intra-individual variation While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype (C) 2010 Elsevier B.V. All rights reserved.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which mag. result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division, A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells, Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus, We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus. (C) 1997 by Radiation Research Society

Long-term genomic instability in human lymphocytes induced by single-particle irradiation

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that ran be delivered, In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing at particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha -particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype, These data suggest that instability may be important in situations where even a single particle traverses human cells. (C) 2001 by Radiation Research Society.

Genomic instability in human lymphocytes irradiated with individual charged particles: Involvement of tumor necrosis factor a in irradiated cells but not bystander cells

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. Ra-222), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of He-3(2+) ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups ( greater than or equal totwofold over controls) and was comparable whether cells were traversed by one or two He-3(2+) ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer. (C) 2005 by Radiation Research Society.

BRCA1 Deficiency Exacerbates Estrogen-Induced DNA Damage and Genomic Instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptora- negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability.We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. © 2014 American Association for Cancer Research.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Deregulated estrogen metabolism in BRCA1 deficient cells induces chromosomal instability.

Objectives: Germline mutations in BRCA1 predispose carriers to a high
incidence of breast and ovarian cancers. The BRCA1 protein functions to maintain
genomic stability via important roles in DNA repair, transcriptional regulation, and
post-replicative repair. Despite functions in processes essential in all cells, BRCA1
loss or mutation leads to tumours predominantly in estrogen-regulated tissues.
Here, we aim to determine if endogenous estrogen metabolites may be an initiator
of genomic instability in BRCA1 deficient cells.

Methods: We analysed DNA DSBs by ?H2AX, 53BP1, and pATM1981
foci and neutral comet assay, estrogen metabolite concentrations by LC-MS/MS,
and BRCA1 transcriptional regulation of metabolism genes by ChIP-chip, ChIP,
and qRT-PCR.

Results: We show that estrogen metabolism is perturbed in BRCA1 deficient
cells resulting in elevated production of 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), and decreased production of the protective metabolite
4-methoxyestradiol. We demonstrate that 2-OHE2 and 4-OHE2 treatment leads
to DNA double strand breaks (DSBs) in breast cells, and these DSBs were exacerbated
in both BRCA1 depleted cells and BRCA1 heterozygous cells (harbouring
185delAG mutation). Furthermore, the DSBs were not repaired efficiently in either
BRCA1 depleted or heterozygous cells, and we found that 2-OHE2 and 4-OHE2
treatment generates chromosomal aberrations in BRCA1 depleted cells. We suggest
that the increase in DNA DSBs in BRCA1 deficient cells is due to loss of
both BRCA1 transcriptional repression of estrogen metabolising genes (such as
CYP1A1 and CYP3A4) and loss of transcriptional activation of detoxification
genes (such as COMT).

Conclusions: We suggest that BRCA1 loss results in estrogen driven tumourigenesis
through a combination of increased expression of estrogen metabolising
enzymes and reduced expression of protective enzymes, coupled with a defect in
the repair of DNA DSBs induced by endogenous estrogen metabolites. The overall
effect being an exacerbation of genomic instability in estrogen regulated tissues in
BRCA1 mutation carriers.

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.