[show abstract][hide abstract] ABSTRACT: Concerned about the risks of mammography screening in the adult population, we analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. Our study shows that an X-ray dose of 20 mGy, which is the standard dose received by the breast surface per two-view mammogram X-ray exploration, induces increased frequencies of DNA double-strand breaks to in vitro aged-but not to young-human mammary epithelial cells. We provide evidence that aged epithelial breast cells are more radiosensitive than younger ones. Our studies point to an inefficient damage response of aged cells to low-dose radiation, this being due to both delayed and incomplete mobilization of repair proteins to DNA strand breaks. This inefficient damage response is translated into an important delay in double-strand break disappearance and consequent accumulation of unrepaired DNA breaks. The result of this is a significant increase in micronuclei frequency in the in vitro aged mammary epithelial cells exposed to doses equivalent to a single mammogram X-ray exploration. Since our experiments were carried out in primary epithelial cell cultures in which cells age at the same time as they undergo replication-dependent telomere shortening, we needed to determine the contribution of these two factors to their phenotype. In this paper, we report that the exogenous expression of human telomerase retrotranscriptase in late population doubling epithelial cells does not rescue its delayed repair phenotype. Therefore, retarded DNA break repair is a direct consequence of cellular aging itself, rather than a consequence of the presence of dysfunctional telomeres. Our findings of long-lasting double strand breaks and incomplete DNA break repair in the in vitro aged epithelial cells are in line with the increased carcinogenic risks of radiation exposures at older ages revealed by epidemiologic studies.
PLoS ONE 01/2013; 8(5):e63052. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International Journal of Molecular Sciences 01/2013; 14(8):15810-26. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy.
[show abstract][hide abstract] ABSTRACT: The interplay between ATM and DNA-PKcs kinases during double strand breaks (DSBs) resolution is still a matter of debate. ATM and DNA-PKcs participate differently in the DNA damage response pathway (DDR), but important common aspects are indeed found: both of them are activated when faced with DSBs, they share common targets in the DDR and the absence of either kinase results in faulty DSB repair. Absence of ATM translates into timely repair that, nevertheless, is incomplete. On the other hand, DNA-PKcs absence translates into slower repair, which in turn gives rise to the accumulation of simple and complex reorganizations. These outcomes confirm that the function of both protein kinases is essential to guarantee genome integrity. Interestingly, V(D)J and CSR recombination events provide a powerful tool to study the interplay between both kinases in DSB repair. Although the physiological DSBs generated during V(D)J and CSR recombination are resolved by the non-homologous end-joining (NHEJ) repair pathway, ATM absence during these events translates into chromosome translocations. These results suggest that NHEJ accuracy is threatened in the absence of ATM, which may play a role in avoiding illegitimate repair by favouring the joining of the correct DNA ends. Indeed, simultaneous DNA-PKcs and ATM deficiency during V(D)J and CSR recombination translates into a synergistic increase in potentially dangerous chromosomal translocations and deletions. Although the exact nature of their interaction remains elusive, the evidence indicates that ATM and DNA-PKcs play complementary roles that allow complete and legitimate DSB repair to be reached. Faithful repair can only be achieved by the presence and correct functioning of both kinases: while DNA-PKcs ensures fast rejoining, ATM guarantees complete repair.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2012; · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nucleoprotein complexes that cap the very ends of the eukaryotic chromosomes, named telomeres, are indispensable for cell viability. Telomeric DNA shortens in each cell division until it cannot exert end-protective functions in human somatic cells. Additionally, several proteins have been described to play a key role in telomere homeostasis preventing chromosome extremities to be recognized as double-stranded breaks (DSBs). When telomeres become dysfunctional, either through excessive shortening or due to defects in the proteins that form its structure, they trigger p53/pRb pathways what limits proliferative lifespan. Impairment of telomere function together with a compromised senescence/apoptosis response leads to chromosome instability. Fusions between dysfunctional telomeres or even between dysfunctional telomeres and DSBs can initiate breakage-fusion-bridge (BFB) cycles. Initially, telomere fusions were proposed to cause only structural abnormalities. Nevertheless, changes in chromosome number have also emerged as a possible consequence of alterations in end capping. Here we review the main aspects of telomeres and telomere-based chromosome instability, highlighting why they have been proposed as a driving force for tumourigenesis.
Frontiers in Bioscience 01/2012; 17:2181-96. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Organisms are continuously exposed to DNA damaging agents, consequently, cells have developed an intricate system known as the DNA damage response (DDR) in order to detect and repair DNA lesions. This response has to be rapid and accurate in order to keep genome integrity. It has been observed that the condensation state of chromatin hinders a proper DDR. However, the condensation state of chromatin is not the only barrier to DDR. In this review, we have collected data regarding the presence of DDR factors on micronuclear DNA lesions that indicate that micronuclei are almost incapable of generating an effective DDR because of defects in their nuclear envelope. Finally, considering the recent observations about the reincorporation of micronuclei to the main bulk of chromosomes, we suggest that, under certain circumstances, micronuclei carrying DNA damage might be a source of chromosome instability.
International Journal of Molecular Sciences 01/2012; 13(9):11569-83. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/2011; 729(1-2):35-40. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Most solid tumors are unable to maintain the stability of their genomes at the chromosome level. Indeed, cancer cells display highly rearranged karyotypes containing translocations, amplifications, deletions, and gains and losses of whole chromosomes, which reshuffle steadily. This chromosomal instability most likely occurs early in the development of cancer, and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease. Different mechanisms may underlie chromosome instability in cancer cells, but a prominent role for telomeres, the tip of linear chromosomes, has been determined. Telomeres are ribonucleoprotein structures that prevent natural chromosome ends being recognized as DNA double-strand breaks, by adopting a loop structure. Loss of telomere function appears from either alteration on telomere-binding proteins or from the progressive telomere shortening that normally occurs under physiological conditions in the majority of cells in tissues. Importantly, unmasked telomeres may either trigger the senescent phenotype that has been linked to the aging process or may initiate the chromosome instability needed for cancer development, depending on the integrity of the DNA damage checkpoint responses. Telomere dysfunction contributes to chromosome instability through end-to-end chromosome fusions entering breakage-fusion-bridge (BFB) cycles. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements, and whole chromosome aneuploidy. Noteworthy is the fact that telomere length heterogeneity among individuals may directly influence the scrambling of the genome at tumor initiation. However, reiterated BFB cycles would randomly reorganize the cell karyotype, thus increasing the genetic diversity that characterizes tumor cells. Even though a direct link is still lacking, multiple evidence lead one to believe that telomere dysfunction directly contributes to cancer development in humans. The expansion of highly unstable cells due to telomere dysfunction enhances the genetic diversity needed to fuel specific mutations that may promote cell immortalization and the acquisition of a tumor phenotype.
Advances in Cancer Research 01/2011; 112:11-41. · 6.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromosomal instability occurs early in the development of cancer and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease. Telomere erosion is one of the factors that contribute to chromosome instability through end-to-end chromosome fusions entering BFB (breakage-fusion-bridge) cycles. Uncapped chromosomes with short dysfunctional telomeres represent an initiating substrate for both pre- and post-replicative joining, which leads to unstable chromosome rearrangements prone to bridge at mitotic anaphase. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements and whole chromosome aneuploidy. Accordingly, telomere-driven instability generates highly unstable genomes that could promote cell immortalization and the acquisition of a tumour phenotype.
Biochemical Society Transactions 12/2010; 38(6):1698-703. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Errors in chromosome segregation during mitosis result in aneuploidy, which in humans may play a role in the onset of neoplasia by changing gene dosage. Nearly all solid tumors exhibit genomic instability at the chromosomal level, showing both structural and numerical chromosome abnormalities. Chromosomal instability occurs early in the development of cancer and may represent an important step in the initiation and/or progression of the disease. Telomere integrity appears to be a critical element in the genesis of structural chromosome imbalances, but it is still not clear whether it can also generate numerical chromosome aberrations. We investigated the possible relationship between telomere shortening and aneuploidy formation in human mammary epithelial cells using the cytokinesis-block micronucleus assay combined with fluorescent DNA probes. In this cell system, uncapped chromosomes fuse with each other resulting in dicentric chromosomes, which are known to be a source of new structural chromosome rearrangements. Here, we show that in primary epithelial cells, the chromosomes with short telomeres are more frequently involved in missegregation events than chromosomes of normal telomere length. Whole chromosome aneuploidy occurs through both nondisjunction and anaphase lagging of dicentric chromatids, which suggests that pulling anaphase bridges toward opposite poles can generate the necessary force for detaching a chromosome from the microtubules of one or both spindle poles. Therefore, telomere-driven instability can promote not only the appearance of chromosomal rearrangements but also the appearance of numerical chromosome aberrations that could favor cell immortalization and the acquisition of a tumor phenotype.
Genes Chromosomes and Cancer 04/2010; 49(4):368-78. · 3.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Micronuclei are good markers of genotoxic exposure in humans and their scoring has been extensively used to identify potential genotoxic agents. Micronuclei are also indicators of chromosomal instability, since the frequency of micronuclei is higher in tumour cells and cells with a defective DNA damage repair system or disrupted cell cycle checkpoint machinery. Despite the widespread use of this biomarker, information on the basic biology of micronuclei and the impact of micronuclei on the cell is relatively controversial. In some cell systems, micronuclei are considered to be genetic material that is lost for the cell; whereas other studies suggest that micronuclear DNA is actively transcribed and its genes are fully expressed. Recently, evidence has accumulated suggesting that damaged DNA entrapped in micronuclei induces a defective cell cycle checkpoint arrest and DNA repair response, and that micronuclear content can be degraded without inducing an immediate cell cycle arrest or causing the cell to enter apoptosis. Overall, these findings emphasise the important consequences of micronucleus formation in terms of chromosomal instability in general and gene loss in particular.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/2010; 705(1):60-7. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome damage relevant to cancer. Although it was initially developed to measure micronuclei, it is also useful for measuring nucleoplasmic bridges and nuclear buds. Abnormal nuclear morphologies are frequently observed in malignant tissues and short-term tumour cell cultures. Changes in chromosome structure and number resulting from chromosome instability are important factors in oncogenesis. Telomeres have become key players in the initiation of chromosome instability related to carcinogenesis by means of breakage-fusion-bridge cycles. To better understand the connection between telomere dysfunction and the appearance of abnormal nuclear morphologies, we have characterised the presence of micronuclei, nucleoplasmic bridges and nuclear buds in human mammary primary epithelial cells. These cells can proliferate beyond the Hayflick limit by spontaneously losing expression of the p16(INK4a) protein. Progressive telomere shortening leads to the loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter into breakage-fusion-bridge cycles generating massive chromosomal instability. In human mammary epithelial cells, different types of abnormal nuclear morphologies were observed, however only nucleoplasmatic bridges and buds increased significantly with population doublings. Fluorescent in situ hybridisation using centromeric and painting specific probes for chromosomes with eroded telomeres has revealed that these chromosomes are preferentially included in the different types of abnormal nuclear morphologies observed, thus reflecting their common origin. Accordingly, real-time imaging of cell divisions enabled us to determine that anaphase bridge resolution was mainly through chromatin breakage and the formation of symmetric buds in daughter nuclei. Few micronuclei emerged in this cell system thus validating the scoring of nucleoplasmic bridges and nuclear buds for measuring chromosome instability in telomere-dysfunction cell environments.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2009; 683(1-2):16-22. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Micronuclei are good markers of chromosome instability and, among other disturbances, are closely related to double-strand break induction. The ability of DNA lesions sequestered in the micronuclear bodies to activate the complex damage-signalling network is highly controversial since some repair factors have not been consistently detected inside micronuclei. In order to better understand the efficiency of the response induced by micronuclear DNA damage, we have analyzed the presence of DNA damage-response factors and DNA degradation markers in these structures. Radiation-induced DNA double-strand breaks produce a modification of chromatin structural proteins, such as the H2AX histone, which is rapidly phosphorylated around the break site. Strikingly, we have been able to distinguish two different phosphoH2AX (gammaH2AX) labelling patterns in micronuclei: discrete foci, indicating DSB presence, and uniform labelling affecting the whole micronucleus, pointing to genomic DNA fragmentation. At early post-irradiation times we observed a high fraction of micronuclei displaying gammaH2AX foci. Co-localization experiments showed that only a small fraction of the DSBs in micronuclei were able to properly recruit the p53 binding protein 1 (53BP1) and the meiotic recombination 11 (MRE11). We suggest that trafficking defects through the micronuclear envelope compromise the recruitment of DNA damage-response factors. In contrast to micronuclei displaying gammaH2AX foci, we observed that micronuclei showing a gammaH2AX extensive-uniform labelling were more frequently observed at substantial post-irradiation times. By means of TUNEL assay, we proved that DNA degradation was carried out inside these micronuclei. Given this scenario, we propose that micronuclei carrying a non-repaired DSB are conduced to their elimination, thus favouring chromosome instability in terms of allele loss.
DNA repair 09/2009; 8(10):1225-34. · 4.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: After irradiation, ATM defective cells accumulate unrepaired double strand breaks (DSBs) for several cell divisions. At the chromosome level, unresolved DSBs appear as chromosome breaks that can be efficiently scored by using telomeric and mFISH probes. H2AX is immediately activated by ATM in response to DNA damage and its phosphorylated form, gammaH2AX, flanks the DSB through several megabases. The gammaH2AX-labeling status of broken chromosome ends was analyzed in AT cells to check whether the DNA damage response was accurately taking place in these persistent DSBs. The results show that one quarter of the scored breaks are devoid of gammaH2AX foci in metaphase spreads from ATM-deficient cells, and this fraction is significantly higher than in normal cells (chi(2) < 0.05). Accumulation of sensor and repair proteins at damaged sites is a key event in the cellular response to DSBs, so MRE11 labeling at broken ends was also analyzed. While all gammaH2AX foci scored at visible broken ends colocalize with MRE11 foci, all gammaH2AX-unlabeled breaks are also devoid of MRE11-labeling. The present results suggest that a significant subset of the AT long-lived DSBs may persist as "invisible" DSBs due to deficient detection by the DNA damage repair machinery. Eventually the properly signaled DSBs will be repaired while invisible breaks may indefinitely accumulate; most probably contributing to the AT cells' well known genomic instability.
Genes Chromosomes and Cancer 06/2009; 48(9):745-59. · 3.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epidemiological studies have demonstrated age differences among human adults in susceptibility to radiation, with cancer cases attributable to radiation being more frequent for older individuals at time of exposure. In addition to the notion that susceptibility increases because of progressive decline in DNA monitoring and immunosurveillance, telomere function is now emerging as a new and important factor in modulating cellular and organism sensitivity to ionizing radiation. The link between telomeres and radiosensitivity is well-documented in humans, but the causal events remain elusive. In this paper, it is shown that irradiated human epithelial cells with short dysfunctional telomeres derived from normal mammary gland display elevated DNA damage. An approach identifying the specific chromosomes with critically shortened telomeres in each donor has allowed us to conclude that short dysfunctional telomeres in human epithelial cells join radiation-induced DNA broken ends, thus interfering with their efficient repair. These findings argue against telomeres participating as sensors or transducers of DNA damage, as previously suggested. Rather, our current findings give support to the idea that dysfunctional telomeres, by acting as an additional joining option, reduce the repair fidelity of DNA broken-ends induced by radiation throughout the genome. In the mammary gland, age-dependent telomere attrition due to epithelial turnover, together with the accretion of checkpoint deficiencies, might render the accumulation of short dysfunctional telomeres. This implies that the risks associated with mammography screening could be higher than previously assumed. Our results have the possibility of imprinting a temporal dimension onto radiation sensitivity, namely, that shortened telomeres in aged cells may more easily compromise normal tissue function in the elderly.
[show abstract][hide abstract] ABSTRACT: Chromosomal instability is increasingly appreciated as a key component of tumorigenesis in humans. A combination of abnormal telomere shortening and cell-cycle checkpoint deficiency has been proposed as the initial lesions causing destabilizing chromatin bridges in proliferative cells. We examined the participation of the different types of end-to-end fusions in generating instable karyotypes in non-transformed human breast epithelial cells. We concluded that short dysfunctional telomeres represent an initiating substrate for post-replicative rejoining of sister chromatids and are likely to make an important contribution to the formation of chromosomal rearrangements and the amplification of chromosome arm segments in breast epithelial cells. We propose that there is a chronological order in the participation of the different types of end-to-end fusions in the generation of chromosomal instability. Thus, intrachromosomal post-replicative joining would proceed mainly in the early stages after overcoming growth arrest, when telomere dysfunction is limited and affects only one chromosome end in a cell. The absence of a second substrate for end joining will conduct the cell with the uncapped chromosome to replicate its DNA and fuse the uncapped sister chromatids after replication. Later, since telomeres shorten progressively with each DNA replication round, the uncapping will affect many more chromosome ends, and fusions between the uncapped ends from different chromosomes will be produced. While the fusion of sister chromatids will produce chromosome segment amplification and terminal deletions in the daughter cells, interchromosomal fusion will produce unbalanced rearrangements other than chromosome segment amplifications.
Cytogenetic and Genome Research 02/2008; 122(3-4):315-25. · 1.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human individuals often exhibit important differences in their sensitivity to ionising radiation. Extensive literature links radiation sensitivity with impaired DNA repair which is due to a lack of correct functioning in many proteins involved in DNA-repair pathways and/or in DNA-damage checkpoint responses. Given that ionising radiation is an important and widespread diagnostic and therapeutic tool, it is important to investigate further those factors and mechanisms that underlie individual radiosensitivity. Recently, evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionising radiation, broadening still further the currently complex and challenging scenario.
[show abstract][hide abstract] ABSTRACT: The development of genomic instability is an important step toward generating the multiple genetic changes required for cancer. Telomere dysfunction is one of the factors that contribute to tumorigenesis. Telomeres shorten with each cell division in the absence of telomerase. Human mammary epithelial cells (HMECs) obtained from normal human tissue demonstrate two growth phases. After an initial phase of active growth, HMECs exhibit a growth plateau termed selection. However, some cells can emerge from this growth plateau by spontaneously losing expression of the p16(INK4a) protein. These post-selection HMECs are capable of undergoing an additional 20-50 population doublings in culture. Continued proliferation of these post-selection HMECs leads to further telomere erosion, loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter bridge-fusion-breakage (BFB) cycles, generating massive chromosomal instability before terminating in a population growth plateau termed agonescence. We have found that the chromosome arms carrying the shortest telomeres are those involved in telomere-telomere type rearrangements. In addition, we found that the risk of a particular chromosome being unstable differs between individuals. Most importantly, we identified sister chromatid fusion as a first event in generating genomic instability in HMECs. During post-selection HMEC growth, double strand breaks are generated by both fused chromosomes as well as individual chromosomes with fused chromatids entering BFB cycles. These broken chromosome extremities are able to join other broken ends or eroded telomeres, producing massive chromosomal instability at the later passages of the cell culture. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
Genes Chromosomes and Cancer 01/2006; 44(4):339-50. · 3.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNA-PKcs-/- MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs-/- cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs-/- MEFs than in their wild-type counterparts. Spectral karyotyping-fluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs-/- MEFs. Because DNA-PKcs-/- MEFs show a strong G1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs-/- MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events.
Cancer Research 12/2005; 65(22):10223-32. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the study was to investigate the spectrum and frequencies of chromosome aberrations induced by the exposure of different mouse spermatogenic germ cell stages to ionizing radiation. Male mice were exposed in vivo to X-rays. Chromosome aberrations were analyzed in first- and second-embryonic cleavages obtained from mating irradiated males with nonirradiated females at different periods after radiation exposure. A combination of telomeric and centromeric labeling as well as whole Y chromosome painting was used to characterize the rejoining pattern and the telomere status of the radiation-induced DNA breaks. The frequency of chromosome aberrations observed in eggs fertilized with sperm irradiated at the early spermatid stage was markedly higher than the frequency in eggs fertilized with sperm irradiated at the other spermatogenic stages when reference was made to the chromosome aberrations recovered in early embryos. At the first division postirradiation, distal rejoining of broken chromosome ends (in regard to the position of the centromere) was more frequent than proximal rejoining; thus compound acentric fragments were more frequently observed than dicentric chromosomes. The presence of additional telomere signals at the broken chromosome ends in mouse germ cells and early embryos, compatible with de novo formation of telomeres, was not frequent.
Journal of Radiation Research 10/2004; 45(3):415-22. · 1.45 Impact Factor