in-vitro-evaluation-oncogenic-transformation-human-mammary-epithelial

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Universitat Autònoma de Barcelona

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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.

Breaks invisible to the DNA damage response machinery accumulate in ATM-deficient cells.

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 lesions sequestered in micronuclei induce a local defective-damage response.

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.

Genetic activities in micronuclei: is the DNA entrapped in micronuclei lost for the cell?

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.

Nuclear envelope defects impede a proper response to micronuclear DNA lesions.

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.

Is DNA damage response ready for action anywhere?

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.

Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro.

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. 

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

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. 

Aging and radiation: bad companions.

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. 

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

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.

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

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.

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

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.

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

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.

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

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  genes to obtain partially transformed cells and also with  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.

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

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.

Article	Year
Breaks invisible to the DNA damage response machinery accumulate in ATM-deficient cells. Genes, chromosomes & cancer\| PubMed ID: 19455703	2009
DNA lesions sequestered in micronuclei induce a local defective-damage response. DNA repair\| PubMed ID: 19683478	2009
Genetic activities in micronuclei: is the DNA entrapped in micronuclei lost for the cell? Mutation research\| PubMed ID: 20307686	2010
Nuclear envelope defects impede a proper response to micronuclear DNA lesions. Mutation research\| PubMed ID: 21945242	2012
Is DNA damage response ready for action anywhere? International journal of molecular sciences\| PubMed ID: 23109871	2012
Increased mammogram-induced DNA damage in mammary epithelial cells aged in vitro. PloS one\| PubMed ID: 23667571	2013
Highly sensitive automated method for DNA damage assessment: gamma-H2AX foci counting and cell cycle sorting. International journal of molecular sciences\| PubMed ID: 23903043	2013
Aging and radiation: bad companions. Aging cell\| PubMed ID: 25645467	2015
Breast primary epithelial cells that escape p16-dependent stasis enter a telomere-driven crisis state. Breast cancer research : BCR\| PubMed ID: 26758019	2016
Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis. BioMed research international\| PubMed ID: 27057549	2016
Impaired nuclear functions in micronuclei results in genome instability and chromothripsis. Archives of toxicology\| PubMed ID: 27542123	2016
Distinct Sets of lncRNAs are Differentially Modulated after Exposure to High and Low Doses of X Rays. Radiation research\| PubMed ID: 27841703	2016
Detection of Impaired DNA Replication and Repair in Micronuclei as Indicators of Genomic Instability and Chromothripsis. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29564826	2018
Radiation-Induced Malignant Transformation of Preneoplastic and Normal Breast Primary Epithelial Cells. Molecular cancer research : MCR\| PubMed ID: 30651374	2019
Identification of reference genes for RT-qPCR data normalisation in aging studies. Scientific reports\| PubMed ID: 31562345	2019

Mariona Terradas

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