Co-occurrences of polymorphic heterochromatin regions of chromosomes and effect on reproductive failure.

Although the polymorphic heterochromatin regions of chromosomes (heteromorphisms) have been extensively studied for their phenotypic effects on humans, co-occurrences of chromosome 1, 9, 16 and Y heteromorphisms and of acrocentric variants have never been studied on humans with an objective scoring system. Here we compared the frequencies of individual heteromorphisms on a total of 602, 768 and 224 patients with the indications of infertility, recurrent miscarriage and in vitro fertilization (IVF) failure, respectively and on 272 controls. Then we examined whether there were significant co-occurrences between heteromorphisms within and between the groups. There were no statistically significant differences in the frequencies of heteromorphisms between the groups. Both statistically significant and non-significant correlations were observed within the non-acrocentric and certain acrocentric heteromorphisms in each group. When these co-occurrences were examined between the groups, a 2.2 fold increased risk of IVF failure in males in the presence of either chromosome 13 or chromosome 21 variants was observed (95 %CI:1.1-4.2). We conclude that the simultaneous manifestations of heteromorphisms have no effect on reproductive failure. There seems to be a correlation between the non-acrocentric heteromorphisms (1qh+, 9qh+, 16qh + and Yqh+/-), which might be the result of complex interactions of formation of these heterochromatin regions. The correlations observed between certain acrocentric chromosomes might be related to satellite association and nucleolus formation. The increased risk observed in males with IVF failure in the presence of either chromosome 13 or 21 variants should be interpreted cautiously due to the heterogeneity of the group.

Assessment of Radio-Induced Damage in Endothelial Cells Irradiated with 40 kVp, 220 kVp, and 4 MV X-rays by Means of Micro and Nanodosimetric Calculations.

The objective of this work was to study the differences in terms of early biological effects that might exist between different X-rays energies by using a mechanistic approach. To this end, radiobiological experiments exposing cell monolayers to three X-ray energies were performed in order to assess the yields of early DNA damage, in particular of double-strand breaks (DSBs). The simulation of these irradiations was set in order to understand the differences in the obtained experimental results. Hence, simulated results in terms of microdosimetric spectra and early DSB induction were analyzed and compared to the experimental data. Human umbilical vein endothelial cells (HUVECs) were irradiated with 40, 220 kVp, and 4 MV X-rays. The Geant4 Monte Carlo simulation toolkit and its extension Geant4-DNA were used for the simulations. Microdosimetric calculations aiming to determine possible differences in the variability of the energy absorbed by the irradiated cell population for those photon spectra were performed on 10,000 endothelial cell nuclei representing a cell monolayer. Nanodosimetric simulations were also carried out using a computation chain that allowed the simulation of physical, physico-chemical, and chemical stages on a single realistic endothelial cell nucleus model including both heterochromatin and euchromatin. DNA damage was scored in terms of yields of prompt DSBs per Gray (Gy) and per giga (109) base pair (Gbp) and DSB complexity was derived in order to be compared to experimental data expressed as numbers of histone variant H2AX (γ-H2AX) foci per cell. The calculated microdosimetric spread in the irradiated cell population was similar when comparing between 40 and 220 kVp X-rays and higher when comparing with 4 MV X-rays. Simulated yields of induced DSB/Gy/Gbp were found to be equivalent to those for 40 and 220 kVp but larger than those for 4 MV, resulting in a relative biological effectiveness (RBE) of 1.3. Additionally, DSB complexity was similar between the considered photon spectra. Simulated results were in good agreement with experimental data obtained by IRSN (Institut de radioprotection et de sûreté nucléaire) radiobiologists. Despite differences in photon energy, few differences were observed when comparing between 40 and 220 kVp X-rays in microdosimetric and nanodosimetric calculations. Nevertheless, variations were observed when comparing between 40/220 kVp and 4 MV X-rays. Thanks to the simulation results, these variations were able to be explained by the differences in the production of secondary electrons with energies below 10 keV.

Paradoxical association of TET loss of function with genome-wide DNA hypomethylation.

Cancer genomes are characterized by focal increases in DNA methylation, co-occurring with widespread hypomethylation. Here, we show that TET loss of function results in a similar genomic footprint. Both 5hmC in wild-type (WT) genomes and DNA hypermethylation in TET-deficient genomes are largely confined to the active euchromatic compartment, consistent with the known functions of TET proteins in DNA demethylation and the known distribution of 5hmC at transcribed genes and active enhancers. In contrast, an unexpected DNA hypomethylation noted in multiple TET-deficient genomes is primarily observed in the heterochromatin compartment. In a mouse model of T cell lymphoma driven by TET deficiency (Tet2/3 DKO T cells), genomic analysis of malignant T cells revealed DNA hypomethylation in the heterochromatic genomic compartment, as well as reactivation of repeat elements and enrichment for single-nucleotide alterations, primarily in heterochromatic regions of the genome. Moreover, hematopoietic stem/precursor cells (HSPCs) doubly deficient for Tet2 and Dnmt3a displayed greater losses of DNA methylation than HSPCs singly deficient for Tet2 or Dnmt3a alone, potentially explaining the unexpected synergy between DNMT3A and TET2 mutations in myeloid and lymphoid malignancies. Tet1-deficient cells showed decreased localization of DNMT3A in the heterochromatin compartment compared with WT cells, pointing to a functional interaction between TET and DNMT proteins and providing a potential explanation for the hypomethylation observed in TET-deficient genomes. Our data suggest that TET loss of function may at least partially underlie the characteristic pattern of global hypomethylation coupled to regional hypermethylation observed in diverse cancer genomes, and highlight the potential contribution of heterochromatin hypomethylation to oncogenesis.

Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity.

How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin.

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

DNA damaging agents trigger the expression of the HML silent mating type locus in Saccharomyces cerevisiae.

Many DNA damaging agents also react with RNA and protein, and could thus cause epigenetic as well as genotoxic changes. To investigate which DNA damaging agents alter epigenetic states, we studied the chemical-induced changes in expression of the yeast silent mating type locus HMLα, which can be triggered by inhibiting yeast Sir2. We observed that the alkylating agent methyl methane sulfonate (MMS) can result in HMLα expression, using a colony sector assay that results from expression of a HML-positioned cre gene. Using single-cell imaging we also observed that alkylating agents, including MMS and methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as short-wave UV, also decreased HML silencing. We suggest that chemical-induced alterations in heterochromatin structure could confer transient phenotypic changes that affect the cellular responses to DNA damaging agents.

Heterochromatin protects retinal pigment epithelium cells from oxidative damage by silencing p53 target genes.

Oxidative stress (OS)-induced retinal pigment epithelium (RPE) cell apoptosis is critically implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Heterochromatin, a compact and transcriptional inert chromatin structure, has been recently shown to be dynamically regulated in response to stress stimuli. The functional mechanism of heterochromatin on OS exposure is unclear, however. Here we show that OS increases heterochromatin formation both in vivo and in vitro, which is essential for protecting RPE cells from oxidative damage. Mechanistically, OS-induced heterochromatin selectively accumulates at p53-regulated proapoptotic target promoters and inhibits their transcription. Furthermore, OS-induced desumoylation of p53 promotes p53-heterochromatin interaction and regulates p53 promoter selection, resulting in the locus-specific recruitment of heterochromatin and transcription repression. Together, our findings demonstrate a protective function of OS-induced heterochromatin formation in which p53 desumoylation-guided promoter selection and subsequent heterochromatin recruitment play a critical role. We propose that targeting heterochromatin provides a plausible therapeutic strategy for the treatment of AMD.

Effect of heterochromatin stability on intestinal stem cell aging in Drosophila.

Chromatin change is one of the crucial causes of aging. Specifically, maintenance of heterochromatin stability is critical for cellular integrity, and its loss induces genomic instability and cellular aging. However, the causes and effects of heterochromatin instability in multicellular tissue aging still remain unclear. Here, in the adult Drosophila midgut, we report age-related loss of heterochromatin stability in enterocytes (ECs) due to the loss and dispersion of tri-methylated histone H3 Lys9 (H3K9me3) and heterochromatin protein 1 (HP1). Our study further shows that EC-specific knockdown of Su(var)3-9, histone lysine methyltransferase for H3K9me3 formation, or HP1a leads to intestinal stem cell (ISC) aging through genomic stress, JNK signaling, and apoptotic death in ECs. Our findings revealed the plausible causes of age-related loss of heterochromatin stability in ECs, including oxidative stress and nutrient-sensing AKT/TOR signaling. Taken together, the loss of heterochromatin stability may be the crucial niche aging mechanism for ISC aging which is the prime determinant of intestinal tissue aging. Furthermore, our study provides new clues on the link between heterochromatin and aging.

Untangling the genetics from the epigenetics in pancreatic cancer metastasis.

Comparative genomic analyses of primary tumors and metastases within individuals with pancreatic cancer have exposed the complex clonal dynamics that underlie the dissemination of cancer cells to distant sites. Recent studies implicate non-genetic mechanisms in this process, particularly fluctuations in chromatin states and metabolism, which can endow rare cells within a primary tumor with metastatic potential.

Pharmacological inhibition of arginine and lysine methyltransferases induces nuclear abnormalities and suppresses angiogenesis in human endothelial cells.

Posttranslational modifications of histone tails can alter chromatin structure and regulate gene transcription. While recent studies implicate the lysine/arginine protein methyltransferases in the regulation of genes for endothelial metabolism, the role of AMI-1 and AMI-5 compounds in angiogenesis remains unknown. Here, we show that global inhibition of arginine and lysine histone methyltransferases (HMTs) by AMI-5 induced an angiostatic profile in human microvascular endothelial cells and human umbilical vein endothelial cells. Based on FACS analysis, we found that inhibition of HMTs significantly affects proliferation of endothelial cells, by suppressing cell cycle progression in the G0/G1 phase. Immunofluorescent studies of the endothelial cells replication pattern by 5-ethynyl-2'-deoxyuridine incorporation disclosed that AMI-5, and the arginine methyltransferase inhibitor AMI-1, induced heterochromatin formation and a number of nuclear abnormalities, such as formation of micronuclei (MNs) and nucleoplasmic bridges (NPBs), which are markers of chromosomal instability. In addition to the modification of the cell cycle machinery in response to AMIs treatment, also endothelial cells migration and capillary-like tube formation processes were significantly inhibited, implicating a stimulatory role of HMTs in angiogenesis.

Vitamin C alleviates aging defects in a stem cell model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.

PRC2 and SWI/SNF Chromatin Remodeling Complexes in Health and Disease.

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.

Histone demethylase JARID1C inactivation triggers genomic instability in sporadic renal cancer.

Mutations in genes encoding chromatin-remodeling proteins are often identified in a variety of cancers. For example, the histone demethylase JARID1C is frequently inactivated in patients with clear cell renal cell carcinoma (ccRCC); however, it is largely unknown how JARID1C dysfunction promotes cancer. Here, we determined that JARID1C binds broadly to chromatin domains characterized by the trimethylation of lysine 9 (H3K9me3), which is a histone mark enriched in heterochromatin. Moreover, we found that JARID1C localizes on heterochromatin, is required for heterochromatin replication, and forms a complex with established players of heterochromatin assembly, including SUV39H1 and HP1α, as well as with proteins not previously associated with heterochromatin assembly, such as the cullin 4 (CUL4) complex adaptor protein DDB1. Transcription on heterochromatin is tightly suppressed to safeguard the genome, and in ccRCC cells, JARID1C inactivation led to the unrestrained expression of heterochromatic noncoding RNAs (ncRNAs) that in turn triggered genomic instability. Moreover, ccRCC patients harboring JARID1C mutations exhibited aberrant ncRNA expression and increased genomic rearrangements compared with ccRCC patients with tumors endowed with other genetic lesions. Together, these data suggest that inactivation of JARID1C in renal cancer leads to heterochromatin disruption, genomic rearrangement, and aggressive ccRCCs. Moreover, our results shed light on a mechanism that underlies genomic instability in sporadic cancers.

Expression of the genetic suppressor element 24.2 (GSE24.2) decreases DNA damage and oxidative stress in X-linked dyskeratosis congenita cells.

The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.

Dynamics of radiation induced γH2AX foci in chromatin subcompartments of mouse pachytene spermatocytes and round spermatids.

Chromatin compaction is thought to influence the severity of radiation-induced DNA damage. We assessed how chromatin state affects DNA double-strand break repair within eu-/heterochromatin domains in male germ cells by profiling the spatiotemporal dynamics of γ-radiation-induced γH2AX foci in confocal images of mouse pachytene spermatocytes and round spermatids (5 min to 16 hr post-irradiation, in vivo). In unirradiated cells, all DNA-dense heterochromatin domains showed compaction by anti-H3K9me3-staining, except for peripheral areas. Following irradiation, this signal was lost within 5 min, but regained later (8-16 hr); these two events coincided with the appearance and loss of γH2AX foci, respectively. While euchromatin showed a large number of bright foci in both cell types, heterochromatin had few foci. In spermatids, a few small, faint foci appeared within chromocenters. Pachytene-stage, on the other hand, lacked foci within heterochromatin, although a few were closely associated with the heterochromatin periphery. The number of euchromatin foci in spermatids showed a dose-dependent enhancement following irradiation (0.5-4 Gy), although no significant increase was seen in the quantity of heterochromatin foci. While all foci in pachytene-stage cells were resolved, spermatids showed large residual foci-especially from heterochromatin foci, which remained faint for up to 4 hr, then increased in size between 8-16 hr, expanding at the chromocenter periphery and eventually protruding into euchromatin at H3K9me3-signal-free areas. Thus, this study identified scant foci formation and poor repair within heterochromatin, with distinctly different dynamics in meiotic and post-meiotic stages of spermatogenesis, and provides direct evidence for heterochromatin decompaction following DNA damage, which facilitates repair/repositioning of foci towards euchromatin domains. It is the first demonstration of spatiotemporal mobilization of double-strand breaks with respect to chromatin subdomains in male germ cells.

Both gender and concurrent chronic lymphocytic thyroiditis may influence the nuclear texture of papillary thyroid carcinomas cells.

A disparity in gender incidence has been reported in both papillary thyroid carcinoma (PTC) and chronic lymphocytic thyroiditis (CLT) diseases frequently associated and whose incidence has been increasing in parallel. We aimed to analyze differences in morphometric variables between male and female PTC patients and their relationship with the presence of concurrent CLT. The nuclear texture features of 100 hematoxylin-eosin stained nuclei from 100 consecutive classic PTC patients enrolled in our service were compared with their clinical and pathological features, including the presence of CLT. All patients were submitted to a standard management protocol and followed-up for 13-248 months (Mo = 117 months). Chromatin in women tended to present a denser and more homogeneous structure, in a less mottled pattern, with higher values of energy (p = 0.008) and diagonal moment (p = 0.032) than men. Concurrent CLT was more prevalent in women (41.42%) than in men (13.33%, p = 0.04) and was associated with higher cluster prominence values (p = 0.027), a parameter that indicates a predominance of high nuclear contrasted heterochromatin. A multivariate logistic regression analysis showed that higher cluster prominence was independently correlated with chromatin in patients who presented CLT but did not demonstrate any association between concurrent CLT and gender. We were unable to demonstrate any association between gender and any characteristic of tumor aggressiveness or patients outcome. Our results suggest that chromatin texture of hematoxylin-eosin stained nuclei in paraffin sections of PTC cells is related to both gender and concurrent CLT.

Mitogen-activated protein kinase p38 and retinoblastoma protein signalling is required for DNA damage-mediated formation of senescence-associated heterochromatic foci in tumour cells.

DNA-damaging agents are able to induce irreversible cell growth arrest and senescence in some types of tumour cells, thus contributing to the static feature of cancer. However, senescent tumour cells may re-enter the cell cycle, leading to tumour relapse. Understanding the mechanisms that control the viability of senescent cells may be critical for tumour suppression. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which contribute to the stability of the senescent state. However, it is unclear whether SAHF formation is universal in tumour cells. We report that the DNA-damaging agents doxorubicin and 7-ethyl-10-hydroxycamptothecin were able to induce the formation of SAHF in some tumour cell types, and this induction was accompanied by activation of the retinoblastoma protein pathway. By contrast, tumour cells in which the retinoblastoma protein pathway could not be activated by doxorubicin or 7-ethyl-10-hydroxycamptothecin failed to form SAHF. In parallel, tumour cells with deficient retinoblastoma protein were also unable to form SAHF. In addition, we show that the mitogen-activated protein kinase p38 pathway was involved in tumour cell SAHF formation in response to doxorubicin and 7-ethyl-10-hydroxycamptothecin. Furthermore, HMG box transcription factor 1 (HBP1), a downstream target of the mitogen-activated protein kinase p38-mediated senescence pathway, was required for SAHF formation. Taken together, the results of the present study highlight the roles of the mitogen-activated protein kinase p38/retinoblastoma protein pathway in tumour cell SAHF formation in response to DNA-damaging agents, and provide new insights into the mechanisms of DNA damage-mediated tumour suppression.

Thiazolidinediones cause compaction of nuclear heterochromatin in the pluripotent mesenchymal cell line C3H10T1/2 when inducing an adipogenic phenotype.

OBJECTIVE: To characterize the nuclear changes induced in vitro by thiazolidinediones (TZDs) in a murine pluripotent mesenchymal cell line. STUDY DESIGN: The C3H10T1/2 cell line, which can differentiate either in osteoblast or in adipocyte, was cultured in the presence of pioglitazone (5 microM) or rosiglitazone (0.5 microM) for 6, 8 and 9 days (D). Quantitative real-time polymerase chain reaction analysis evaluated the expression of key genes of the adipocytic or osteoblastic differentiation (PPARgamma[peroxisome proliferatoractivated receptor gamma], Runx2 [runt-related transcription factor 2] and alkaline phosphatase). Cells were stained with Oil Red O for lipids, and chromatin was counter-stained with hematoxylin. Cells were photographed at x 1,000 magnification and analyzed with texture analysis software. Nuclear area, mean gray level and run-length parameters were calculated. RESULTS: PPARgamma was significantly expressed from D6 (normalized ratio > 7) in TZD groups (ratio >27 at D9). No significant differences were found for either Runx2 or alkaline phosphatase expression versus control at D6 or D9. Cells cultured with TZDs began to differentiate into adipocytes with numerous lipid droplets which appeared at D6. Nuclear area decreased suddenly at D6 for both TZDs, and the mean gray level increased. Run-length parameters changed significantly due to chromatin compaction. CONCLUSION: TZDs provoked differentiation of C3H10T1/2 into adipocytes, leading to inactivation of genes that were highly compacted into heterochromatin.

Nuclear morphometry, nucleomics and prostate cancer progression.

Prostate cancer (PCa) results from a multistep process. This process includes initiation, which occurs through various aging events and multiple insults (such as chronic infection, inflammation and genetic instability through reactive oxygen species causing DNA double-strand breaks), followed by a multistep process of progression. These steps include several genetic and epigenetic alterations, as well as alterations to the chromatin structure, which occur in response to the carcinogenic stress-related events that sustain proliferative signaling. Events such as evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis are readily observed. In addition, in conjunction with these critical drivers of carcinogenesis, other factors related to the etiopathogenesis of PCa, involving energy metabolism and evasion of the immune surveillance system, appear to be involved. In addition, when cancer spread and metastasis occur, the 'tumor microenvironment' in the bone of PCa patients may provide a way to sustain dormancy or senescence and eventually establish a 'seed and soil' site where PCa proliferation and growth may occur over time. When PCa is initiated and progression ensues, significant alterations in nuclear size, shape and heterochromatin (DNA transcription) organization are found, and key nuclear transcriptional and structural proteins, as well as multiple nuclear bodies can lead to precancerous and malignant changes. These series of cellular and tissue-related malignancy-associated events can be quantified to assess disease progression and management.

Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients.