Role of H4K16 acetylation in 53BP1 recruitment to double-strand break sites in in vitro aged cells.

Increased frequency of DNA double strand breaks (DSBs) with aging suggests an age-associated decline in DSB repair efficiency, which is also influenced by the epigenetic landscape. H4 acetylation at lysine 16 (H4K16Ac) has been related to DSB repair since deacetylation of this mark is required for efficient 53BP1 recruitment to DSBs. Although age-associated changes in H4K16Ac levels have been studied, their contribution to age-related DSB accumulation remains unknown. In vitro aged Human Dermal Fibroblasts (HDFs) display lower levels of H4K16A that correlate with reduced recruitment of 53BP1 to basal DSBs. Following DNA damage induction, early passage (EP) cells suffered from a transient H4K16 deacetylation that allowed proper 53BP1 recruitment to DSBs. In contrast, to reach this specific and optimum level, aged cells responded by increasing their overall lower H4K16Ac levels. Induced hyperacetylation of late passage (LP) cells using trichostatin A increased H4K16Ac levels but did not ameliorate 53BP1 recruitment. Instead, deacetylation induced by MOF silencing reduced H4K16Ac levels and compromised 53BP1 recruitment in both EP and LP cells. Age-associated decrease of H4K16Ac levels contributes to the repair defect displayed by in vitro aged cells. H4K16Ac responds to DNA damage in order to reach a specific, optimum level that allows proper 53BP1 recruitment. This response may be compromised with age, as LP cells depart from lower H4K16Ac levels. Variations in H4K16Ac following the activation of the DNA damage response and aging point at this histone mark as a key mediator between DNA repair and age-associated chromatin alterations.

A matter of wrapper: Defects in the nuclear envelope of lagging and bridging chromatin threatens genome integrity.

The nuclear envelope surrounds the eukaryotic genome and, through the nuclear pore complexes, regulates transport in and out of the nucleus. Correct nucleo-cytoplasm compartmentations are essential for nuclear functions such as DNA replication or repair. During metazoan mitosis, the nuclear envelope disintegrates to allow the segregation of the two copies of DNA between daughter cells. At the end of mitosis, it reforms on each group of chromosomes in the daughter cells. However, nuclear envelope reformation is delayed on lagging chromosomes and DNA bridges. Defects in the coordination between nuclear envelope reformation and chromosome segregation impair the nuclear functions. Mechanical stress to which micronuclei and DNA bridges are subjected to combined with their particular architecture and the altered nuclear functions result in DNA damage. While micronuclei and DNA bridges were considered for more than 100 years as mere indicators of chromosomal instability, rapid technological advances are helping to better understand the biological consequences of these aberrant nuclear morphologies. Recent studies provide interesting evidence that micronuclei and chromatin bridges act as a key platforms for a catastrophic mutational process observed in cancers called chromothripsis and a trigger for the innate immune response. Therefore, they could affect cellular functions by both genetic and non-genetic means.

Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells.

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3'OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

Age-associated deficient recruitment of 53BP1 in G1 cells directs DNA double-strand break repair to BRCA1/CtIP-mediated DNA-end resection.

DNA repair mechanisms play a crucial role in maintaining genome integrity. However, the increased frequency of DNA double-strand breaks (DSBs) and genome rearrangements in aged individuals suggests an age-associated DNA repair deficiency. Previous work from our group revealed a delayed firing of the DNA damage response in human mammary epithelial cells (HMECs) from aged donors. We now report a decreased activity of the main DSB repair pathways, the canonical non-homologous end-joining (c-NHEJ) and the homologous recombination (HR) in these HMECs from older individuals. We describe here a deficient recruitment of 53BP1 to DSB sites in G1 cells, probably influenced by an altered epigenetic regulation. 53BP1 absence at some DSBs is responsible for the age-associated DNA repair defect, as it permits the ectopic formation of BRCA1 foci while still in the G1 phase. CtIP and RPA foci are also formed in G1 cells from aged donors, but RAD51 is not recruited, thus indicating that extensive DNA-end resection occurs in these breaks although HR is not triggered. These results suggest an age-associated switch of DSB repair from canonical to highly mutagenic alternative mechanisms that promote the formation of genome rearrangements, a source of genome instability that might contribute to the aging process.

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells.

Tumorigenesis is a multi-step process in which cells acquire capabilities that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions. A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay. Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

Identification of reference genes for RT-qPCR data normalisation in aging studies.

Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.

Delayed γH2AX foci disappearance in mammary epithelial cells from aged women reveals an age-associated DNA repair defect.

Aging is a degenerative process in which genome instability plays a crucial role. To gain insight into the link between organismal aging and DNA repair capacity, we analyzed DNA double-strand break (DSB) resolution efficiency in human mammary epithelial cells from 12 healthy donors of young and old ages. The frequency of DSBs was measured by quantifying the number of γH2AX foci before and after 1Gy of γ-rays and it was higher in cells from aged donors (ADs) at all times analyzed. At 24 hours after irradiation, ADs retained a significantly higher frequency of residual DSBs than young donors (YDs), which had already reached values close to basal levels. The kinetics of DSB induction and disappearance showed that cells from ADs and YDs repair DSBs with similar speed, although analysis of early times after irradiation indicate that a repair defect may lie within the firing of the DNA repair machinery in AD cells. Indeed, using a mathematical model we calculated a constant factor of delay affecting aged human epithelial cells repair kinetics. This defect manifests with the accumulation of DSBs that might eventually undergo illegitimate repair, thus posing a relevant threat to the maintenance of genome integrity in older individuals.

Radiation-Induced Malignant Transformation of Preneoplastic and Normal Breast Primary Epithelial Cells.

Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. IMPLICATIONS: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.

Detection of Impaired DNA Replication and Repair in Micronuclei as Indicators of Genomic Instability and Chromothripsis.

The physical isolation of chromosomes within micronuclei offers an attractive mechanistic explanation for the local DNA fragmentation and clustered genome rearrangements that characterize chromothripsis. Localized shattering of the chromatin confined in micronuclei can be a consequence of defects in micronuclei basic general functions, such as DNA replication and repair. The detection of DNA repair and replication defects in micronuclei is described here, as well as the analysis of chromosome breakage and inaccurate reassembly of broken segments in the daughter cells, as indirect methods to detect chromothripsis.

Distinct Sets of lncRNAs are Differentially Modulated after Exposure to High and Low Doses of X Rays.

High- and low-dose X rays are used in medicine as therapeutic and diagnostic tools, respectively. While the cellular response to high-dose radiation is well known, studies on the effects of low-dose radiation and its ability to trigger a proper DNA damage response have had contradictory results. The functions of many signaling and effector proteins of the DNA damage response (DDR) have been described, and are attributed to well-known DDR pathways. However, there has been little known about the contribution of long noncoding RNAs (lncRNAs) to DDR, although there is recent evidence that lncRNAs may be associated with almost all biological functions, including DDR. In this work, we investigated the participation of lncRNAs in the response to different X-ray doses. By microarray analysis, we observed that in human breast epithelial cells, distinct sets of coding and noncoding transcripts are differentially regulated after moderate- and high-dose irradiation compared to those regulated after low-dose irradiation. While the modulated coding and noncoding genes at low doses relate to cell signaling pathways, those affected by moderate and high doses are mostly enriched for cell cycle regulation and apoptotic pathways. Quantification using qPCR of the lncRNAs identified by microarrays allowed the validation of 75% of those regulated at the higher doses. These results indicate that lncRNA expression is regulated by ionizing radiation and that this expression is dose dependent.

Impaired nuclear functions in micronuclei results in genome instability and chromothripsis.

Micronuclei (MN) have generally been considered a consequence of DNA damage and, as such, have been used as markers of exposure to genotoxic agents. However, advances in DNA sequencing methods and the development of high-resolution microscopy with which to analyse chromosome dynamics in live cells have been fundamental in building a more refined view of the existing links between DNA damage and micronuclei. Here, we review recent progress indicating that defects of micronuclei affect basic nuclear functions, such as DNA repair and replication, generating massive damage in the chromatin of the MN. In addition, the physical isolation of chromosomes within MN offers an attractive mechanistic explanation for chromothripsis, a massive local DNA fragmentation that produces complex rearrangements restricted to only one or a few chromosomes. When micronuclear chromatin is reincorporated in the daughter cell nuclei, the under-replicated, damaged or rearranged micronuclear chromatin might contribute to genome instability. The traditional conception of micronuclei has been overturned, as they have evolved from passive indicators of DNA damage to active players in the formation of DNA lesions, thus unravelling previously unforeseen roles of micronuclei in the origins of chromosome instability.

Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis.

In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70-85% of the AT viable cells (TUNEL-negative) carried ≥ 10 γH2AX foci/cell, while only 12-27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥ 10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

Chromosome Bridges Maintain Kinetochore-Microtubule Attachment throughout Mitosis and Rarely Break during Anaphase.

Accurate chromosome segregation during cell division is essential to maintain genome stability, and chromosome segregation errors are causally linked to genetic disorders and cancer. An anaphase chromosome bridge is a particular chromosome segregation error observed in cells that enter mitosis with fused chromosomes/sister chromatids. The widely accepted Breakage/Fusion/Bridge cycle model proposes that anaphase chromosome bridges break during mitosis to generate chromosome ends that will fuse during the following cell cycle, thus forming new bridges that will break, and so on. However, various studies have also shown a link between chromosome bridges and aneuploidy and/or polyploidy. In this study, we investigated the behavior and properties of chromosome bridges during mitosis, with the idea to gain insight into the potential mechanism underlying chromosome bridge-induced aneuploidy. We find that only a small number of chromosome bridges break during anaphase, whereas the rest persist through mitosis into the subsequent cell cycle. We also find that the microtubule bundles (k-fibers) bound to bridge kinetochores are not prone to breakage/detachment, thus supporting the conclusion that k-fiber detachment is not the cause of chromosome bridge-induced aneuploidy. Instead, our data suggest that while the microtubules bound to the kinetochores of normally segregating chromosomes shorten substantially during anaphase, the k-fibers bound to bridge kinetochores shorten only slightly, and may even lengthen, during anaphase. This causes some of the bridge kinetochores/chromosomes to lag behind in a position that is proximal to the cell/spindle equator and may cause the bridged chromosomes to be segregated into the same daughter nucleus or to form a micronucleus.

Breast primary epithelial cells that escape p16-dependent stasis enter a telomere-driven crisis state.

Breast cancer is the most common malignant disease in women, but some basic questions remain in breast cancer biology. To answer these, several cell models were developed. Recently, the use of improved cell-culture conditions has enabled the development of a new primary cell model with certain luminal characteristics. This model is relevant because, after the introduction of a specific set of genetic elements, the transformed cells yielded tumors resembling human adenocarcinomas in mice. The use of improved cell-culture conditions supporting the growth of these breast primary epithelial cells was expected to delay or eliminate stress-induced senescence and lead to the propagation of normal cells. However, no studies have been carried out to investigate these points. Propagation of breast primary epithelial cells was performed in WIT medium on Primaria plates. Immunofluorescence, western blot and qRT-PCR were used to detect molecular markers, and to determine the integrity of DNA damage-response pathways. Promoter methylation of p16 (INK4a) was assessed by pyrosequencing. In order to obtain a dynamic picture of chromosome instability over time in culture, we applied FISH methodologies. To better link chromosome instability with excessive telomere attrition, we introduced the telomerase reverse transcriptase human gene using a lentiviral vector. We report here that breast primary epithelial cells propagated in vitro with WIT medium on Primaria plates express some luminal characteristics, but not a complete luminal lineage phenotype. They undergo a p16-dependent stress-induced senescence (stasis), and the cells that escape stasis finally enter a crisis state with rampant chromosome instability. Chromosome instability in these cells is driven by excessive telomere attrition, as distributions of chromosomes involved in aberrations correlate with the profiles of telomere signal-free ends. Importantly, ectopic expression of the human TERT gene rescued their chromosomal instability phenotype. Essentially, our data show that contrary to what was previously suggested, improved culture conditions to propagate in vitro mammary epithelial cells with some luminal characteristics do not prevent stress-induced senescence. This barrier is overcome by spontaneous methylation of the p16 (INK4a) promoter, allowing the proliferation of cells with telomere dysfunction and ensuing chromosome instability.

Centrosome aberrations in human mammary epithelial cells driven by cooperative interactions between p16INK4a deficiency and telomere-dependent genotoxic stress.

Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.

Genomic instability: Crossing pathways at the origin of structural and numerical chromosome changes.

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.

Aging and radiation: bad companions.

Aging involves a deterioration of cell functions and changes that may predispose the cell to undergo an oncogenic transformation. The carcinogenic risks following radiation exposure rise with age among adults. Increasing inflammatory response, loss of oxidant/antioxidant equilibrium, ongoing telomere attrition, decline in the DNA damage response efficiency, and deleterious nuclear organization are age-related cellular changes that trigger a serious threat to genomic integrity. In this review, we discuss the mechanistic interplay between all these factors, providing an integrated view of how they contribute to the observed age-related increase in radiation sensitivity. As life expectancy increases and so it does the medical intervention, it is important to highlight the benefits of radiation protection in the elderly. Thus, a deep understanding of the mechanistic processes confining the threat of aging-related radiosensitivity is currently of foremost relevance.

γH2AX foci on apparently intact mitotic chromosomes: not signatures of misrejoining events but signals of unresolved DNA damage.

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci-regardless their coincidence with a visible break-always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.

Telomere-dependent genomic integrity: evolution of the fusion-bridge-breakage cycle concept.

Most cancer genomes show abnormalities in chromosome structure and number, two types of aberrations that could share a common mechanistic origin through proliferation-dependent loss of telomere function. Impairment of checkpoints that limit cell proliferation when telomeres are critically short might allow unrestrained cell division. The resulting uncapped chromosomes can fuse to each other, forming unstable configurations that can bridge during mitosis. Chromatin bridges can break to generate new broken ends that will then fuse with other broken ends. Successive events of break and fusion will continuously generate unbalanced chromosomal rearrangements, leading to gene-copy gains and losses. However, chromosome bridges do not always break. Evidence has recently been obtained to suggest that telomere-dependent chromosome bridges remaining unbroken can hinder cytokinesis and yield tetraploid cells. This might constitute an unstable intermediate in tumorigenesis, as progressive losses of individual chromosomes due to geometrical defects during cell division result in subtetraploid karyotypes. Additionally, the presence of short dysfunctional telomeres in cells can also cause these cells to become sensitive to mutagens, and particularly to radiation exposure. Human individuals exhibit differences in their sensitivity to radiation, which can be relevant for choice of therapy. Telomere function may well be involved in cellular and organism responses to ionizing radiation. Since eroded telomeres are sensed and act as double-strand breaks, they can interact with radiation-induced breaks, sharply increasing the possibility of misjoining. Altogether, this scenario provides certain clues to understanding the important role of telomeres in maintaining genomic integrity.

Highly sensitive automated method for DNA damage assessment: gamma-H2AX foci counting and cell cycle sorting.

Phosphorylation of the H2AX protein is an early step in the double strand break (DSB) repair pathway; therefore, phosphorylated histone (γH2AX) foci scoring is widely used as a measure for DSBs. Foci scoring is performed either manually or semi-automatically using hand-operated capturing and image analysis software. In general, both techniques are laborious and prone to artifacts associated with manual scoring. While a few fully automated methods have been described in the literature, none of them have been used to quantify γH2AX foci in combination with a cell cycle phase analysis. Adding this feature to a rapid automated γH2AX foci quantification method would reduce the scoring uncertainty that arises from the variations in the background level of the γH2AX signal throughout the cell cycle. The method was set up to measure DNA damage induced in human mammary epithelial cells by irradiation under a mammogram device. We adapted a FISH (fluorescent in situ hybridization) Spot-counting system, which has a slide loader with automatic scanning and cell capture system throughout the thickness of each cell (z-stack), to meet our assay requirements. While scanning the sample, the system classifies the selected nuclei according to the signal patterns previously described by the user. For our purposes, a double staining immunofluorescence was carried out with antibodies to detect γH2AX and pericentrin, an integral component of the centrosome. We could thus distinguish both the number of γH2AX foci per cell and the cell cycle phase. Furthermore, restrictive settings of the program classifier reduced the "touching nuclei" problem described in other image analysis software. The automated scoring was faster than and as sensitive as its manually performed counterpart. This system is a reliable tool for γH2AX radio-induced foci counting and provides essential information about the cell cycle stage. It thus offers a more complete and rapid assessment of DNA damage.