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DNA methylation (DNAm) changes are of increasing relevance to neurodegenerative disorders, including Huntington's disease (HD). We performed genome-wide screening of possible DNAm changes occurring during striatal differentiation in human induced pluripotent stem cells derived from a HD patient (HD-hiPSCs) as cellular model. We identified 240 differentially methylated regions (DMRs) at promoters in fully differentiated HD-hiPSCs. Subsequently, we focused on the methylation differences in a subcluster of genes related to Jumonji Domain Containing 3 (JMJD3), a demethylase that epigenetically regulates neuronal differentiation and activates neuronal progenitor associated genes, which are indispensable for neuronal fate acquisition. Noticeably among these genes, WD repeat-containing protein 5 (WDR5) promoter was found hypermethylated in HD-hiPSCs, resulting in a significant down-modulation in its expression and of the encoded protein. A similar WDR5 expression decrease was seen in a small series of HD-hiPSC lines characterized by different CAG length. The decrease in WDR5 expression was particularly evident in HD-hiPSCs compared to hESCs and control-hiPSCs from healthy subjects. WDR5 is a core component of the MLL/SET1 chromatin remodeling complexes essential for H3K4me3, previously reported to play an important role in stem cells self-renewal and differentiation. These results suggest the existence of epigenetic mechanisms in HD and the identification of genes, which are able to modulate HD phenotype, is important both for biomarker discovery and therapeutic interventions.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system. GBM is a fatal tumor, incurable by conventional therapies. One of the factors underlying tumor recurrence and poor long-term survival is the presence of a cancer stem-like cell population, termed glioma stem cells (GSCs), which is particularly resistant to chemotherapy and radiotherapy and supports tumor self-renewal. The aim of the present study was to evaluate the impact and difference in effects of short-term and long-term treatments with valproic acid (VPA), a histone deacetylase inhibitor, on seven GSC lines. We investigated for the first time the changes in the genome-wide DNA methylation profile and the differentiation behavior of GSCs induced by short-term and long-term VPA treatments. Moreover, we verified VPA sensitivity after long-term VPA pretreatment and, notably, the results provide evidence of a subpopulation more resistant to further VPA treatments. Finally, since short-term VPA treatment induced a reversal of the MGMT methylation status, we aimed to sensitize GSCs to temozolomide, the drug commonly used for this tumor, using this regimen. The overall data highlighted the heterogeneous behavior of GSC lines that is representative of tumor heterogeneity in GBM. The VPA effects were variable among these cell lines in terms of pro-differentiating ability and DNA methylation switch. Here, we attempted to identify a suitable therapy for the eradication of the stem cell subpopulation, which is mandatory to achieve an effective treatment for this tumor. Differentiation-inducing and epigenetic therapies are the most promising approaches to affect the multiple properties of GSCs and, finally, defeat GBM.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and the most common malignant brain tumor. Current therapies provide a median survival of 12–15 months after diagnosis, due to the high recurrence rate. The failure of current therapies may be due to the presence, within the tumor, of cells characterized by enhanced self-renewal capacity, multilineage differentiation potential and elevated invasive behavior, called glioma stem cells (GSCs). To evaluate the pharmacological efficacy of selected drugs on six GSC lines, we set up a multiple drug responsivity assay based on the combined evaluation of cytomorphological and functional parameters, including the analysis of polymorphic nuclei, mitotic index and cell viability. In order to understand the real pharmacological efficacy of the tested drugs, we assigned a specific drug responsivity score to each GSC line, integrating the data produced by multiple assays. In this work we explored the antineoplastic effects of paclitaxel (PTX), an inhibitor of microtubule depolymerization, utilized as standard treatment in several cancers, and of valproic acid (VPA), an inhibitor of histone deacetylases (HDACs) with multiple anticancer properties. We classified the six GSC lines as responsive or resistant to these drugs, on the basis of their responsivity scores. This method can also be useful to identify the best way to combine two or more drugs. In particular, we utilized the pro-differentiating effect of VPA to improve the PTX effectiveness and we observed a significant reduction of cell viability compared to single treatments.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term "multiforme" describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common "signature" of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomic-signature (gain of chromosome 7- EGFR gene, loss of chromosome 13- RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes. This new overview could have a huge importance in therapy.

Introduction. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods. In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on hBM-MSC malignant transformation in the literature.

The importance of the genetic factor in the aetiology of premature ovarian failure (POF) is emphasized by the high percentage of familial cases and X chromosome abnormalities account for 10% of chromosomal aberrations. In this study, we report the detailed analysis of 4 chromosomal abnormalities involving the X chromosome and associated with POF that were detected during a screening of 269 affected women. Conventional and molecular cytogenetics were valuable tools for locating the breakpoint regions and thus the following karyotypes were defined: 46,X,der(X)t(X;19)(p21.1;q13.42)mat, 46,X,t(X;2)(q21.33;q14.3)dn, 46,X,der(X)t(X;Y)(q26.2;q11.223)mat and 46,X,t(X;13)(q13.3;q31)dn. A bioinformatic analysis of the breakpoint regions identified putative candidate genes for ovarian failure near the breakpoint regions on the X chromosome or on autosomes that were involved in the translocation event. HS6ST1, HS6ST2 and MATER genes were identified and their functions and a literature review revealed an interesting connection to the POF phenotype. Moreover, the 19q13.32 locus is associated with the age of onset of the natural menopause. These results support the position effect of the breakpoint on flanking genes, and cytogenetic techniques, in combination with bioinformatic analysis, may help to improve what is known about this puzzling disorder and its diagnostic potential.

BACKGROUND. Glioblastoma multiforme (GBM) is the most common and malignant type of glioma and it is characterized by extensive heterogeneity, both at the cellular and molecular level. The poor prognosis and the lack of an effective treatment are due to the presence of a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). At the genomic level, heterogeneity is characterized by multiple levels of alterations, including cytogenetic, genomic and epigenomic alterations. Drug sensitivity is an additional level of GSC complexity and heterogeneity and the identification of an effective treatment for GBM depends on the depletion of the GSC pool. Valproic acid (VPA) is a histone deacetylase inhibitor and so it can be used for an epigenetic therapy for cancer. Besides, a differentiation inducing ability of VPA on cancer cells was demonstrated. Paclitaxel (PTX) is a conventional chemotherapeutic agent and in the last years it was shown to be a potential therapeutic drug for gliomas. AIMS AND PROJECT DESIGN. Six GSC lines were studied, as they represent a valuable tool for the investigation of cytogenomic and epigenomic landscapes of GBM, in order to unravel specific molecular pathways, involved in the stem-like counterpart. Drug sensitivity profiles were assessed, evaluating cell viability and cytomorphological parameters (mitotic index, ploidy and polymorphic nuclei), after VPA and PTX administration. The reliability of differentiation and epigenetic therapy through the use of VPA was further investigated by morphological and molecular epigenomic analysis, investigating the DNA methylation status. RESULTS AND DISCUSSION. Several shared cytogenetic and genomic alterations linked to GBM pathogenesis were found among the GSC lines. Specifically, polysomy of chromosome 7, loss of chromosome 10, CDKN2A and CDKN2B deletions are aberrations related to highly relevant pathways in GBM tumorigenesis. Moreover, a minimal deleted region at 1p36.31 was common among the six GSC lines, including CAMTA1 gene, a putative tumor suppressor gene, specific for cancer stem-like cells. Disregulated cytogenenomic pathways in GSCs were preferentially linked to the control of stem cell proliferation, invasion, cellular development and differentiation. The evaluation of the methylation profiles of GSC lines revealed aberrant methylation of developmental genes, which are targeted by Polycomb Repressive Complex 2 in embryonic stem cells and involved in cellular development and nervous system differentiation, evidencing a specific impairment of these processes in cancer stem-like cells. VPA is able to begin a differentiation process in GSCs, as demonstrated by the study of methylation changes caused by VPA, through the methylation of pathways which are involved in self-renewal maintenance, such as Wnt/β-catenin, and several cancer-related mechanisms. Anyway, terminal differentiation was impaired, due to an intrinsic characteristic of cancer cells endowed with stem like properties. GSCs viability was severely affected by dual drug treatment, combining VPA and PTX: VPA caused an initial differentiation, enabling PTX to induce cell death of downstream cells in tumor hierarchy. Thus, a dual approach with drugs affecting different features of malignancy could be a successful approach to GBM treatment. CONCLUSIONS. A multi-level study for the evaluation of cytogenomic and epigenomic landscapes of GSCs is an effective approach for the identification of molecular pathways, specifically de-regulated in stem-like cells, giving an outstanding contribution in the identification of key mechanisms sustaining self-renewal. GSC lines are a valuable tool to evaluate the potentiality of new therapeutical approaches, which should be able to overwhelm the stem-like related counterpart. VPA and PTX combined treatment was found to fulfill the therapeutical potential of VPA and might be a successful approach to unlock the self-renewal loop, typical of GSCs and affect their growth.

Transitional cell carcinoma (TCC) comprises the majority of bladder cancers accounting for more than 90%. In general, TCC is grouped in high- or low-grade (HG or LG), noninvasive (NI) or invasive (IN) lesions. The biological differences between these groups probably reflect underlying genetic heterogeneity leading to specific pathways of tumor development and progression. Twenty-two TCCs (nine HGIN, three HGNI, one LGIN, nine LGNI) were tested by UroVysion® Bladder Cancer Kit, a fluorescence in situ hybridization assay designed to detect aneuploidy for chromosomes 3, 7, 17 and loss of the 9p21 locus; the test was performed in duplicate on formalin-fixed paraffin-embedded (FFPE) samples and on freshly isolated nuclei (FIN) in order to evaluate the performance of this targeted test in two different materials derived from the same tumor. Although FFPE specimens offer the certainty of histological diagnosis and allow retrospective studies of a large number of sample, conversely fresh tissues are the most reliable for molecular genetic analysis and also provide a comprehensive analysis of the biopsy. At least 100 cells for each preparation were scored and the signals divided according to loss, disomy and gain (number of signals/cell

Th e capability of a tumor to grow and propagate seems to depend on a small sub-population of cells, called cancer stem cells (CSCs) [1]. We isolated and characterized putative bladder CSCs populations from primary Transitional Cell Carcinomas (TCCs). Th ese cells proliferated as urospheres and had abilities for extensive proliferation and self-renewal. Their positivity for stem cell markers and their potential to diff erentiate were assessed. Urospheres gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might refl ect their progressive loss of self-renewal ability. Conventional cytogenetic analysis was carried out on fresh chromosome spreads immediately aft er the isolation and aft er one week of culture. Th e data indicated important karyotype selection and the loss of complexity present in fresh tumors. Another purpose was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of TCCs [2], on Formalin Fixed Paraffi n Embedded (FFPE) tissues and interphasic nuclei of urospheres: comparing tissue and aft er-culture data, we observed a great heterogeneity also among samples with the same histotype. The comparison between array-CGH and UroVysion assay evidenced the same heterogeneity on two different types of material derived from the same tumor: FFPE and Freshly Isolated interphasic Nuclei (FIN). Our results confi rmed the importance of use complementary techniques such array-CGH and FISH, as the former is able to detect alterations at the genome level, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions.

Glioblastoma multiforme (GBM) is one of the most malignant brain tumors and is characterized by multiple genomic alterations such as chromosomal rearrangements and DNA copy number variations. Recent genome-wide studies show a remarkable genomic heterogeneity in GBM and the importance of giving an insight in cancer genome. We analyzed a small subpopulation of cells within the tumor, called cancer stem cells (CSCs), that are able to initiate and sustain tumor growth and recurrence. In particular, we analyzed five cancer stem-like cell lines from GBM to conventional cytogenetics (QFQ banding) and molecular cytogenetics techniques (FISH array, Human Genome CGH microarray kit 44K, Agilent Technologies). This approach allows evaluating recurrent cytogenetic and genomic alterations and also new aberrations in order to identify novel putative loci involved in GBMpathogenesis. The evaluation of chromosome number confirmed tumor heterogeneity as the modal number varied from near-diploid to tetraploid and allowed investigating cell line stability in vitro. Moreover, the karyotype analysis identified the most common chromosomal aberrations in GBM, such as polisomy of chromosome 7 (5/5) and loss of chromosome 10 (3/5). Molecular karyotype confirmed the conventional cytogenetic data and revealed, within the genomic heterogeneity, the presence of specific copy number alterations (CNAs). These CNAs map in loci usually associated with peculiar pathways involved in GBMpathogenesis: gain of whole chromosome 7 (5/5) and amplification of EGFR, complete loss of 9p21.3 (CDKN2A and CDKN2B genes, 4/5), pseudomonosomy for whole chromosome 10 loss (3/5), gain of 1q32.1 (MDM4, 2/5) and amplification of PDGFR gene (4q12, 2/5). This methodological approach allowed integrating different dimensions in genome study and delineating the major aberrations in the cytogenetic and genomic profiles of CSC from GBM

The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well-known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low-level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha-satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low-level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause.

Emerging evidence in the literature suggests the presence of a small subset of cells within tumors, called cancer stem cells (CSCs), that drive tumor formation and growth and are responsible for tumor recurrence. Long-lived stem cells are strongly exposed to genotoxic stresses and, therefore, are prone to the accumulation of different mutations, a key factor in carcinogenesis. Brain tumor stem cells have been identified and isolated from different types of brain tumors, including glioblastoma multiforme (GBM), one of the most common and malignant tumors of central nervous system. GBM is characterized by resistance to surgical resection, radiotherapy, and chemotherapy. In fact, conventional therapies may reduce the tumor mass by killing mainly cells with limited proliferative potential (the “bulk tumor”), but they do not kill CSCs that are able to re-generate new tumors. Therefore it would be interesting to study the mechanism that controls CSCs from the genetic point of view, in order to design new targeted therapeutic approaches restricted to stem cells. In this study we carried out a molecular cytogenetic analysis on two CSC lines isolated from a patient affected by GBM, named GBM2 and GBM7. In particular the karyotypes were evaluated using conventional cytogenetic analysis through QFQ-banding. Then the molecular karyotypes were investigated by array CGH using a Human Genome CGH microarray kit 44K (Agilent Technologies) to underline similarities and differences between the two lines. In particular we found in both lines polyploidy for chromosome 7 (more than four copies) and a complete loss of the region 9p21.1-21.3, which includes the CDKN2A and CDKN2B genes implicated in cell cycle regulatory pathways, including the p53 and retinoblastoma pathways. Therefore, we tested the efficacy of two antineoplastic drugs such as valproic acid (a histone deacetylase inhibitor) and paclitaxel (a microtubule depolymerization inhibitor) assessing, both on drug-treated and untreated cultures, different cytologic and cytogenetics parameters: chromosome number per metaphase, mitotic index, and nuclear morphology. All these data will be compared in order to address future studies on specific genes and to establish guidelines for expression studies and drug testing.

Background Premature ovarian failure (POF) is a secondary hypergonadotrophic amenorrhea occurring before the age of 40 and affecting 1-3% of females. Chromosome anomalies account for 6-8% of POF cases, but only few cases are associated with translocations involving X and Y chromosomes. This study shows the cytogenetic and molecular analysis of a POF patient came to our attention as she developed a left ovary choriocarcinoma at the age of 10 and at 14 years of age she presented secondary amenorrhea with elevated levels of gonadotropins. Results Breakpoint position on X and Y chromosomes was investigated using Fluorescent In Situ Hybridisation (FISH) with a panel of specific BAC probes, microsatellite analysis and evaluation of copy number changes and loss of heterozigosity by Affymetrix® GeneChip platform (Santa Clara, CA, USA). Patient's karyotype resulted 46, X, der(Y)t(X;Y)(q13.1;q11.223). X inactivation study was assessed by RBA banding and showed preferential inactivation of derivative chromosome. The reciprocal spatial disposition of sexual chromosome territories was investigated using whole chromosome painting and centromeres probes: patient's results didn't show a significant difference in comparison to normal controls. Conclusion The peculiar clinical case come to our attention highlighted the complexity of POF aetiology and of the translocation event, even if our results seem to exclude any effect on nuclear organisation. POF phenotype could be partially explained by skewed X chromosome inactivation that influences gene expression.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs. © 2009 Macmillan Publishers Limited. All rights reserved.

Bladder cancer is the seventh most common cancer worldwide. Urothelial carcinoma (formerly known as transitional cell carcinoma, TCC) comprises the majority of bladder cancers, accounting for more than 90%. In general, TCC is grouped into high- or low-grade (HG- or LG) lesions, based on their genetic profile and clinical behavior. The majority of bladder primary TCCs are LG (grades I and II) noninvasive (NI, pathologic stage pTa) papillary lesions. TCC is associated with a high rate of recurrence (75% after 5 years), and a distinct rate (10%–30%) of progression to HG lesions (grade III), carcinoma in situ, or invasive (IN, pT1). The UroVysion Bladder Cancer Kit (UroVysion Kit) is a US Food and Drug Administration-approved fluorescence in situ hybridization (FISH) probe set for use in the detection of recurrent urothelial carcinoma and in patients with hematuria. It is a multi-target multi-color assay designed to detect aneuploidies for chromosomes 3, 7, 17, and loss of the 9p21 locus in urine specimens. In this study we applied the UroVysion test on freshly isolated interphase nuclei and paraffin-embedded tissue from 22 TCCs (9 HGIN, 3 HGNI, 1 LGIN, 9 LGNI), in order to compare numerical aberrations in the two cell samples from the same tumor. 100 morphologically abnormal nuclei were considered for each sample in both tests, recording hybridization signals corresponding to each probe. The signals were divided according to loss (number of signals/cell < 2), disomy (number of signals/cell = 2), and gain (number of signals/cell > 3). Although the number of samples is low, we observed a general heterogeneity in the copy number changes, also among samples with the same histotype and, surprisingly, within the same sample comparing the two tests carried out. In particular, in the HGIN and LGNI tumors we found differences in copy numbers of chromosomes 3, 7, and 17 (p < 0.01) in 67%–89% of samples; for the 9p21 locus the differences were less significant. Our data provide further evidence for the intra-tumor heterogeneity present in different subpopulations of the same tumor, with interesting insights for diagnostic purposes.