Pericentromeric Satellite DNA Research Articles

Visual assessment of global chromatin intranuclear localization and its cellular diversification in mouse cells.

DNA is the core that controls the life activities of cells. In eukaryotic cells, DNA is dispersedly packaged into multiple strands of chromatin to regulate cellular life activities, however, the underlying logic and significance of this configuration remain unclear. In mouse cells, pericentromeric satellite DNA from multiple strands of chromatin can aggregate to form chromocenters [1,2]. Based on microscopic imaging techniques and analysis, we further reveal that there is a non-random recombination aggregation between chromatin in different mouse nuclei, leading to differentiation of nuclear structure and correlation with cell function. We speculate that the recombination aggregation of chromatin nuclear localization can further enrich limited DNA genetic information, and its induced nuclear structural diversification is crucial for the formation of cellular diversification.

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFβ, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.

Defective Satellite DNA Clustering into Chromocenters Underlies Hybrid Incompatibility in Drosophila.

Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI) (Yunis and Yasmineh 1971; Henikoff et al. 2001; Ferree and Barbash 2009; Sawamura 2012; Jagannathan and Yamashita 2017), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into “chromocenters,” thereby bundling chromosomes to maintain the entire genome in a single nucleus (Jagannathan et al. 2018, 2019). Here, we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation, and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.

Expansion of rDNA and pericentromere satellite repeats in the genomes of bank voles Myodes glareolus exposed to environmental radionuclides.

Altered copy number of certain highly repetitive regions of the genome, such as satellite DNA within heterochromatin and ribosomal RNA loci (rDNA), is hypothesized to help safeguard the genome against damage derived from external stressors. We quantified copy number of the 18S rDNA and a pericentromeric satellite DNA (Msat‐160) in bank voles (Myodes glareolus) inhabiting the Chernobyl Exclusion Zone (CEZ), an area that is contaminated by radionuclides and where organisms are exposed to elevated levels of ionizing radiation. We found a significant increase in 18S rDNA and Msat‐160 content in the genomes of bank voles from contaminated locations within the CEZ compared with animals from uncontaminated locations. Moreover, 18S rDNA and Msat‐160 copy number were positively correlated in the genomes of bank voles from uncontaminated, but not in the genomes of animals inhabiting contaminated, areas. These results show the capacity for local‐scale geographic variation in genome architecture and are consistent with the genomic safeguard hypothesis. Disruption of cellular processes related to genomic stability appears to be a hallmark effect in bank voles inhabiting areas contaminated by radionuclides.

The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin.

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

CENP-A binding domains and recombination patterns in horse spermatocytes

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this “centromere effect” is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres.

Isolation of a Pericentromeric Satellite DNA Family in Chnootriba argus (Henosepilachna argus) with an Unusual Short Repeat Unit (TTAAAA) for Beetles

Ladybird beetles (Coccinellidae) are one of the largest groups of beetles. Among them, some species are of economic interest since they can act as a biological control for some agricultural pests whereas other species are phytophagous and can damage crops. Chnootriba argus (Coccinellidae, Epilachnini) has large heterochromatic pericentromeric blocks on all chromosomes, including both sexual chromosomes. Classical digestion of total genomic DNA using restriction endonucleases failed to find the satellite DNA located on these heterochromatic regions. Cloning of C0t-1 DNA resulted in the isolation of a repetitive DNA with a repeat unit of six base pairs, TTAAAA. The amount of TTAAAA repeat in the C. argus genome was about 20%. Fluorescence in situ hybridization (FISH) analysis and digestion of chromosomes with the endonuclease Tru9I revealed that this repetitive DNA could be considered as the putative pericentromeric satellite DNA (satDNA) in this species. The presence of this satellite DNA was tested in other species of the tribe Epilachnini and it is also present in Epilachna paenulata. In both species, the TTAAAA repeat seems to be the main satellite DNA and it is located on the pericentromeric region on all chromosomes. The size of this satDNA, which has only six base pairs is unusual in Coleoptera satellite DNAs, where satDNAs usually have repeat units of a much larger size. Southern hybridization and FISH proved that this satDNA is conserved in some Epilachnini species but not in others. This result is in concordance with the controversial phylogenetic relationships among the genera of the tribe Epilachnini, where the limits between genera are unclear.

The modular mechanism of chromocenter formation in Drosophila.

A central principle underlying the ubiquity and abundance of pericentromeric satellite DNA repeats in eukaryotes has remained poorly understood. Previously we proposed that the interchromosomal clustering of satellite DNAs into nuclear structures known as chromocenters ensures encapsulation of all chromosomes into a single nucleus (Jagannathan et al., 2018). Chromocenter disruption led to micronuclei formation, resulting in cell death. Here we show that chromocenter formation is mediated by a 'modular' network, where associations between two sequence-specific satellite DNA-binding proteins, D1 and Prod, bound to their cognate satellite DNAs, bring the full complement of chromosomes into the chromocenter. D1 prod double mutants die during embryogenesis, exhibiting enhanced phenotypes associated with chromocenter disruption, revealing the universal importance of satellite DNAs and chromocenters. Taken together, we propose that associations between chromocenter modules, consisting of satellite DNA binding proteins and their cognate satellite DNA, package the Drosophila genome within a single nucleus.

A conserved function for pericentromeric satellite DNA

A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus. However, the underlying mechanism to ensure such a configuration is unknown. Here, we provide evidence that pericentromeric satellite DNA, which is often regarded as junk, is a critical constituent of the chromosome, allowing the packaging of all chromosomes into a single nucleus. We show that the multi-AT-hook satellite DNA-binding proteins, Drosophila melanogaster D1 and mouse HMGA1, play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into 'chromocenters', a cytological association of pericentromeric heterochromatin. Defective chromocenter formation leads to micronuclei formation due to budding from the interphase nucleus, DNA damage and cell death. We propose that chromocenter and satellite DNA serve a fundamental role in encapsulating the full complement of the genome within a single nucleus, the universal characteristic of eukaryotic cells.

Epigenetic Reprogramming of Pericentromeric Satellite DNA in Premalignant and Malignant Lesions.

Repression of repetitive DNA is important for maintaining genomic stability, but is often perturbed in cancer. For instance, the megabase satellite domain at chromosome 1q12 is a common site of genetic rearrangements, such as translocations and deletions. Polycomb-group proteins can be observed as large subnuclear domains called polycomb bodies, the composition and cellular function of which has remained elusive. This study demonstrates that polycomb bodies are canonical subunits of the multiprotein polycomb repressive complex 1 deposited on 1q12 pericentromeric satellite DNA, which are normally maintained as constitutive heterochromatin by other mechanisms. Furthermore, the data reveal that polycomb bodies are exclusive to premalignant and malignant cells, being absent in normal cells. For instance, polycomb bodies are present in melanocytic cells of nevi and conserved in primary and metastatic melanomas. Deposition of polycomb on the 1q12 satellite DNA in melanoma development correlated with reduced DNA methylation levels. In agreement with this, inhibition of DNA methyltransferases, with the hypomethylating agent guadecitabine (SGI-110), was sufficient for polycomb body formation on pericentromeric satellites in primary melanocytes. This suggests that polycomb bodies form in cancer cells with global DNA demethylation to control the stability of pericentromeric satellite DNA. These results reveal a novel epigenetic perturbation specific to premalignant and malignant cells that may be used as an early diagnostic marker for detection of precancerous changes and a new therapeutic entry point.Implications: Pericentromeric satellite DNA is epigenetically reprogrammed into polycomb bodies as a premalignant event with implications for transcriptional activity and genomic stability. Mol Cancer Res; 16(3); 417-27. ©2018 AACR.

Function of Junk: Pericentromeric Satellite DNA in Chromosome Maintenance.

Satellite DNAs are simple tandem repeats that exist at centromeric and pericentromeric regions on eukaryotic chromosomes. Unlike the centromeric satellite DNA that comprises the vast majority of natural centromeres, function(s) for the much more abundant pericentromeric satellite repeats are poorly understood. In fact, the lack of coding potential allied with rapid divergence of repeat sequences across eukaryotes has led to their dismissal as "junk DNA" or "selfish parasites." Although implicated in various biological processes, a conserved function for pericentromeric satellite DNA remains unidentified. We have addressed the role of satellite DNA through studying chromocenters, a cytological aggregation of pericentromeric satellite DNA from multiple chromosomes into DNA-dense nuclear foci. We have shown that multivalent satellite DNA-binding proteins cross-link pericentromeric satellite DNA on chromosomes into chromocenters. Disruption of chromocenters results in the formation of micronuclei, which arise by budding off the nucleus during interphase. We propose a model that satellite DNAs are critical chromosome elements that are recognized by satellite DNA-binding proteins and incorporated into chromocenters. We suggest that chromocenters function to preserve the entire chromosomal complement in a single nucleus, a fundamental and unquestioned feature of eukaryotic genomes. We speculate that the rapid divergence of satellite DNA sequences between closely related species results in discordant chromocenter function and may underlie speciation and hybrid incompatibility.

Cohesin facilitates zygotic genome activation in zebrafish.

At zygotic genome activation (ZGA), changes in chromatin structure are associated with new transcription immediately following the maternal-to-zygotic transition (MZT). The nuclear architectural proteins cohesin and CCCTC-binding factor (CTCF) contribute to chromatin structure and gene regulation. We show here that normal cohesin function is important for ZGA in zebrafish. Depletion of the cohesin subunit Rad21 delays ZGA without affecting cell cycle progression. In contrast, CTCF depletion has little effect on ZGA, whereas complete abrogation is lethal. Genome-wide analysis of Rad21 binding reveals a change in distribution from pericentromeric satellite DNA and other locations, including the miR-430 locus (the products of which are responsible for maternal transcript degradation), to genes, as embryos progress through the MZT. After MZT, a subset of Rad21 binding overlaps the pioneer factor Pou5f3, which activates early expressed genes. Rad21 depletion disrupts the formation of nucleoli and RNA polymerase II foci, suggestive of global defects in chromosome architecture. We propose that Rad21/cohesin redistribution to active areas of the genome is key to the establishment of chromosome organization and the embryonic developmental program.

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

Normal segregation of a foreign-species chromosome during Drosophila female meiosis despite extensive heterochromatin divergence.

The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. Here we use a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D. melanogaster. These two species differ by ∼12-13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. We find a visible imbalance in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, we find that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females and does not experience elevated nondisjunction. We conclude that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence. Animal chromosomes typically contain large amounts of noncoding repetitive DNA that nevertheless varies widely between species. This variation may potentially induce non-Mendelian transmission of chromosomes. We have examined the meiotic properties and transmission of a highly diverged chromosome 4 from a foreign species within the fruitfly Drosophila melanogaster. This chromosome has substantially less of a simple sequence repeat than does D. melanogaster 4, and we find that this difference results in altered positioning when chromosomes align during meiosis. Yet this foreign chromosome segregates at normal frequencies, demonstrating that chromosome segregation can be robust to major differences in repetitive DNA abundance.

Satellite DNA transcripts in blood plasma as potential markers of tumor growth

Recent studies of human and animal tumor tissues revealed a high transcriptional activity of pericentromeric satellite DNA repeats; i.e., they were found to produce up to half of all transcripts in tumor cells, which is many times more than in normal cells. It was also found that the two subtypes of satellite DNA (HSATII and GSATII) are transcribed reciprocally, i.e., HSATII transcripts strongly dominate in tumors, while GSATII transcripts are more abundant in the corresponding normal tissues. Since different RNAs are present in blood plasma, and some of them serve as efficient tumor markers, this study was undertaken to evaluate for the first time satellite HSATII and GSATII RNA levels in the plasma of healthy donors and cancer patients. The RT-PCR protocol designed for this purpose enabled detection of both HSATII and GSATII transcripts. It was shown that HSATII transcripts were more abundant than GSATII RNAs in the plasma of healthy donors, and this relationship was inverse in the plasma of cancer patients; these ratios were exactly opposite to those that exist within normal and tumor cells. Some notions concerning the probable origins of circulating satellite RNAs and their role as potential tumor markers are discussed.

Organization and evolution of a novel cervid satellite DNA with yeast CDEI-like repeats

AbstractBackgroundIt has been proposed that pericentromeric satellite DNA arises from the progressive proximal expansion of ancient centromeric DNA. In an attempt to recover putative ancestral centromeric DNA, we microdissected the pericentromeric/centromeric DNA from the chromosome X + 3 of Indian muntjac (Muntiacus muntjak vaginalis) and constructed a microclone-library of the X + 3 centromeric DNA.ResultsA new cervid satellite DNA element, designated as satellite VI, was isolated from this library. Fluorescence in situ hybridization (FISH) studies revealed that satellite VI is predominately located on the distal pericentromeric region of the Indian muntjac chromosome X + 3 and on the pericentromeres of several Old World deer species studied. Its sequence is organized as 11-bp monomeric (ATCACGTGGGA) tandem repeats. Further sequencing on a BAC clone of Indian muntjac harboring this repeat showed that an array of this repeat stretches over approximately 5 kb followed by approximately 3 kb of interspersed repetitive sequences, such as long interspersed elements (LINEs), short interspersed elements (SINEs), and long terminal repeats (LTRs).ConclusionsBased on the chromosomal localization, genomic and sequence organization, and copy numbers of satellite VI in deer species studied, we postulate that this newly found satellite DNA could be a putative ancient cervidic centromeric DNA that is still preserved in some Old World deer. Interestingly, the first eight nucleotides of the 11-bp monomeric consensus sequences are highly conserved and identical to the CDEI element in the centromere of the budding yeastSaccharomyces cerevisiae. The centromeric/pericentromeric satellite DNA harboring abundant copies of CDEI sequences is the first found in a mammalian species. Several zipper-like d (GGGA)2motifs were also found in the (ATCACGTGGGA)nrepeat of satellite VI DNA. Whether the satellite VI is structurally and functionally correlated with the CDEI of centromere of budding yeast and whether a zipper-like structure forms in satellite VI require further studies.

Structure and population dynamics of the major satellite DNA in the red flour beetle Tribolium castaneum

In the beetle genus Tribolium, satellite DNAs comprise a significant amount of pericentromeric heterochromatin and are characterized by rapid turnover resulting in species specific profiles. In the present work we characterize the major pericentromeric satellite DNA TCAST of the beetle T. castaneum and analyse its population dynamics. Using direct sequencing of genomic PCR products we show that the TCAST satellite exists in the form of two related subfamilies: Tcast1a and Tcast1b that make up 20 and 15% of the genome, respectively. Tcast1a and Tcast1b have consensus sequences of 377 and 362 bp respectively, share an average similarity of 79% and are characterized by a divergent, subfamily specific region of approximately 100 bp. The two subfamilies are prevalently organized in the interspersed form, although a portion exists in the form of homogenous tandem arrays composed of only Tcast1a or Tcast1b. The pattern of restriction enzyme digestion indicates that Tcast1a and Tcast1b are organized in composite higher order repeats. Comparison of sequence variability of Tcast1a and Tcast1b among ten strains reveals a difference in the frequency of particular mutations present at some positions. However, no difference in the organization and in the amount of subfamilies was detected among strains. The results show that direct genomic sequencing can be a useful method for the detection of population specific features of satellite DNA. In the case of TCAST satellite DNA, changes in the mutational profiles seem to represent the first step in the genesis of a population specific satellite profile.

The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

Satellite DNA Spatial Localization and Transcriptional Activity in Mouse Embryonic E-14 and IOUD2 Stem Cells

The formation of heterochromatin begins in the differentiating cells. The aim of this work was to study changes of satellite DNA distribution, transcriptional activity and interaction with certain proteins in mouse embryonic stem cells after induction with retinoic acid. We found that pericentromeric satellites entered chromocenters only some days after induction of E-14 and IOUD2 mouse embryonic stem cells. The redistribution was accompanied by association with HP1a and transcription from pericentromeric (but not centromeric) satellite DNA. RNA was polyadenylated and transcribed from the forward chain. Probes made from the cDNA hybridized to all chromosomes. In differentiating cells, the transcript was found exclusively in chromocenters while in differentiated cultured L929 cells it formed 1–2 large clusters outside chromocenters. Using ChIP and immunostaining, we demonstrated that in induced cells pericentromeric DNA interacted with RNA-helicase p68 that was previously shown to be involved in transcription regulation and to be involved in differentiation processes.

Chromosomal analysis of Barrett's cells: demonstration of instability and detection of the metaplastic lineage involved

Barrett's esophagus is lined by columnar and goblets cells with gastric and intestinal characteristics. Despite the association between goblet elements and malignancy, it was not demonstrated that other columnar cells lineages are not related to neoplasia. Chromosomal abnormalities were described in metaplasia adjacent to Barrett's neoplasia, but it is unknown which metaplastic lineages are involved. This work assessed the frequency and the type of chromosomal abnormalities in Barrett's esophagus without neoplasia and performed the identification of the metaplastic cells carrying chromosomal gains. Barrett's esophagus biopsies were collected and processed for short-term cell culture and cytogenetic analysis. Combined immunofluorescence/fluorescence in situ hybridization was performed in cases exhibiting chromosomal gains by using antisera against intestinal (MUC2) and gastric (MUC5AC and MUC6) apomucins and chromosome pericentromeric alpha satellite DNA probes for the chromosomes involved. Each case was scored for the number of spots (0, 1, 2, >2) in 200 nonoverlapping nuclei. Columnar and goblet cells were separately assessed. Short-term cell cultures were achieved in 40/60 cases (67%). There were clonal abnormalities in 27/40 cases (68%) and tetraploid (4n) clones in 10/40 (25%). Structural alterations were detected in 14/40 (35%) with recurrent breakpoints at 1q21, 15q15 and 15q22. Numerical changes (trisomies 7 and 18 and loss of Y) occurred in 16/40 (40%). Gains of chromosomes 7 and 18 were more frequent in columnar than in goblet cells (9.8% vs 0.7% (P<0.05)) and (7.9 vs 1.9% (P<0.05)) respectively. These alterations were detected in cells exhibiting gastric as well as intestinal features and were more frequent in cells without apomucin production. Conclusions: (1) chromosomal instability is a common finding in Barrett's esophagus without neoplasia. (2) The two metaplastic populations are committed, chromosomal gains being more frequent in columnar nongoblet than in goblet cells. (3) The two metaplastic phenotypes, gastric and intestinal, are equally involved.