A new, automated, four-colour interphase FISH approach for the simultaneous detection of specific aneuploidies of diagnosis and prognosis significance in high hyperdiploid ALL

In hyperdiploid acute lymphoblastic leukaemia (ALL), the simultaneous occurrence of specific aneuploidies confers a more favourable outcome than hyperdiploidy alone. Interphase (I) FISH complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in non-dividing cells. To overcome the limits of manual I-FISH, we developed an automated four-colour I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10 and 17. Parameters established for automatic nucleus selection and signal detection were evaluated (3 controls). Cut-off values were determined (10 controls, 1000 nuclei/case). Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 ALL patients (1500 nuclei/patient) were compared with those by CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed by I-FISH. I-FISH revealed numerous additional abnormal clones, ranging between 0.1 % and 31.6%, based on the large number of nuclei evaluated. Four-colour automated I-FISH permits the identification of concurrent aneuploidies of prognostic significance in hyperdiploid ALL. Large numbers of cells can be analysed rapidly by this method. Owing to its high sensitivity, the method provides a powerful tool for the detection of small abnormal clones at diagnosis and during follow up. Compared to CC, it generates a more detailed cytogenetic picture, the biological and clinical significance of which merits further evaluation. Once optimised for a given set of probes, the system can be easily adapted for other probe combinations.

Automated four-color interphase fluorescence in situ hybridization approach for the simultaneous detection of specific aneuploidies of diagnostic and prognostic significance in high hyperdiploid acute lymphoblastic leukemia.

In high hyperdiploid acute lymphoblastic leukemia (ALL), the concurrence of specific trisomies confers a more favorable outcome than hyperdiploidy alone. Interphase fluorescence in situ hybridization (FISH) complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in nondividing cells. To overcome the limits of manual I-FISH, we developed an automated four-color I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10, and 17. Parameters established for nucleus selection and signal detection were evaluated. Cutoff values were determined. Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 patient samples were compared with those obtained with CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed also by I-FISH, and I-FISH revealed numerous additional abnormal clones in all patients, ranging from < or =1% to 31.6% of cells analyzed. We conclude that four-color automated I-FISH permits the identification of concurrent aneuploidies of potential prognostic significance. Large numbers of cells can be analyzed rapidly. The large number of nuclei scored revealed a high level of chromosome variability both at diagnosis and relapse, the prognostic significance of which is of considerable clinical interest and merits further evaluation.

Tetrasomy 8 in a patient with acute nonlymphocytic leukemia: a metaphase and interphase study with fluorescence in situ hybridization.

Tetrasomy 8 constitutes a relatively rare recurring chromosome defect in myeloid disorders. The patient reported here, a 71-year-old man, presented with tetrasomy 8 as the sole chromosome abnormality associated with an acute nonlymphocytic leukemia of the M2 type. He failed to respond to chemotherapy and died one year after diagnosis. Following conventional cytogenetics and fluorescence in situ hybridization (FISH) with a centromeric probe specific for chromosome 8, tetrasomy 8 was detected in 61% of the metaphases analyzed and trisomy 8 in 39%. FISH analysis of interphase nuclei confirmed the existence of tetrasomic (35%) and trisomic cells (56%) and revealed a number of cells with two chromosomes 8 (8%). This normal population may represent lymphocytes or myeloid cells that escaped conventional analysis due to their inability to divide or to the small number of metaphases available. The relatively higher proportion of tetrasomic cells in metaphase compared with interphase may be attributed to a proliferative advantage of tetrasomic cells in vitro or to the longer duration of their cell cycle. The simultaneous presence of trisomic and tetrasomic cells confirms the hypothesis of a clonal relationship between trisomy 8 and tetrasomy 8. Our case brings further evidence to the specificity of tetrasomy 8 to myeloid disorders and to the association of this chromosome abnormality with a relatively poor prognosis. However, new patients must be studied to further delineate this cytogenetic entity.

A new, automated, four-colour interphase FISH approach for the simultaneous detection of specific aneuploidies of diagnostic and prognostic significance in high hyperdiploid ALL

In hyperdiploid acute lymphoblastic leukaemia (ALL), the simultaneous occurrence of specific aneuploidies confers a more favourable outcome than hyperdiploidy alone. Interphase (I) FISH complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in non-dividing cells. To overcome the limits of manual I-FISH, we developed an automated four-colour I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10 and 17. Parameters established for automatic nucleus selection and signal detection were evaluated (3 controls). Cut-off values were determined (10 controls, 1000 nuclei/case). Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 ALL patients (1500 nuclei/patient) were compared with those by CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed by I-FISH. I-FISH revealed numerous additional abnormal clones, ranging between 0.1% and 31.6%, based on the large number of nuclei evaluated. Four-colour automated I-FISH permits the identification of concurrent aneuploidies of prognostic significance in hyperdiploid ALL. Large numbers of cells can be analysed rapidly by this method. Owing to its high sensitivity, the method provides a powerful tool for the detection of small abnormal clones at diagnosis and during follow up. Compared to CC, it generates a more detailed cytogenetic picture, the biological and clinical significance of which merits further evaluation. Once optimised for a given set of probes, the system can be easily adapted for other probe combinations.

Models that include supercoiling of topological domains reproduce several known features of interphase chromosomes.

Understanding the structure of interphase chromosomes is essential to elucidate regulatory mechanisms of gene expression. During recent years, high-throughput DNA sequencing expanded the power of chromosome conformation capture (3C) methods that provide information about reciprocal spatial proximity of chromosomal loci. Since 2012, it is known that entire chromatin in interphase chromosomes is organized into regions with strongly increased frequency of internal contacts. These regions, with the average size of ∼1 Mb, were named topological domains. More recent studies demonstrated presence of unconstrained supercoiling in interphase chromosomes. Using Brownian dynamics simulations, we show here that by including supercoiling into models of topological domains one can reproduce and thus provide possible explanations of several experimentally observed characteristics of interphase chromosomes, such as their complex contact maps.

The dynamics of yeast telomeres and silencing proteins through the cell cycle.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.

Clonal heterogeneity and chromosomal instability at disease presentation in high hyperdiploid acute lymphoblastic leukemia.

Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2-44.7%), compared with those in non-HeH ALL (3.2-6.4%), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Human thymus exports naive CD8 T cells that can home to nonlymphoid tissues.

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.

Corneal Opacities in Trisomy 8 Mosaic Syndrome : Clinical, Histologic and Genetic Features

Purpose: To describe the clinical, histologic and genetic findings of corneal opacities in the trisomy 8 mosaic syndrome. Methods: 3 children aged 8 years (Patients A), 6 years (Patients B) and 1 month (Patients C) respectively, were referred with corneal opacities for ophthalmologic evaluation. The 2 older patients had been previously diagnosed with trisomy 8 mosaicism, while the third was diagnosed after the ocular examination. Automated lamellar keratoplasty (ALTK) was performed on the most amblyopic eye. Histopathologic analysis with immunohistochemical markers and cytogenetic studies by FISH using haploid probes for chromosome 8 and chromosome 16 (control) were performed on the excised corneal lesion. Results: All patients presented vascularized corneal opacities involving the superficial stroma, and amblyopia with a bilateral involvement in two of them (Patients A and B). Post-operative follow-up (range 6-20 months) was satisfactory, with the graft remaining clear and improved visual acuity, allowing iso-acuity and stereoscopy in the one month old child (Patients C). The clinically observed corneal opacities corresponded histopathologically to the replacement of the normal anterior corneal stroma by a choristomatous loose richly vascularized connective tissue containing mucopolysacharides. Bowman's membrane was absent. There were no adnexal structures. The overlaying epithelium expressed keratin 3 in all three cases. Keratin 19 was found in the suprabasal epithelial cells in one case but was absent in the other cases. There were no expression of keratin 7 and 1 as well as MUC5AC in the epithelial cells. FISH analysis from 100 interphase cells of the affected tissue and normal conjontival probe revealed normal diploid cells. Conclusions: In this series, the corneal opacities associated with trisomy 8 mosaic syndrome share a common clinical, histopathological and genetic features. ALTK should be considered at diagnosis to prevent amblyopia in these children.

Controlling cytokinesis in the fission yeast Schizosaccharomyces pombe : the role of dma1; and ubc8

ABSTRACT The fission yeast Schizosaccharomyces pombe is a single celled eukaryote that has proved to be an excellent model system for the study of cell cycle control. S. pombe cells are rod shaped and grow mainly by elongation at their tips. They divide by formation of medially-placed cell wall, or septum, which cleaves the cell in two. Once the cell commits itself to mitosis the site of division is determined by formation of an acto-myosin based contractile ring at the cell cortex. The ring is assembled in stages throughout mitosis and contracts at the end of anaphase, coincident with spindle disassembly. The contraction, but not the assembly, of the ring requires the signal transduction network called the septation initiation network or SIN. The core components of the SIN are three protein kinases (cdc7p, sidl p and sid2p) and their regulatory subunits (spg1 p, cdcl4p and moblp, respectively). Signalling is dependent upon the nucleotide status of the GTPase spgl p, which is regulated by a two-component GAP protein, cdc16p-byr4p. Signalling is thought to emanate from the spindle pole body, where core SIN components are anchored to a scaffold comprised of sid4p and cdc11p. Activation of the SIN requires the protein kinase plolp, which also has additional roles in mitosis. SIN signalling is tightly regulated to assure the proper co-ordination of mitosis and cytokinesis. Ectopic activation of the SIN in interphase can uncouple septum formation from mitosis, while deregulated SIN signalling leads to formation of cells with multiple septa that do not cleave. Regulators of SIN activity are therefore of considerable interest. This study has concentrated upon two of these, dma1 and ubc8. I have demonstrated that dmal becomes essential when SIN signalling is activated. This leads me to propose a tripartite model for regulation of the SIN during the mitotic cell cycle. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this thesis. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN Localisation of ubc8p indicates that it is a nuclear protein. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired. These data suggest that it may be an effector of the spindle assembly checkpoint. Together, these data shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. RESUME La levure Schizosaccharomyces pombe est un eucaryote unicellulaire qui est un bon système d'étude du cycle cellulaire. Les cellules de S. pombe sont en forme de bâtonnets et poussent par allongement aux deux bouts. Elles se divisent en formant une paroi au milieu de la cellule, qui s'appelle un septum et qui sépare la cellule en deux. Une fois que la cellule est engagée dans la mitose, le site de clivage est déterminé par la formation d'un anneau contractile d'acto-myosine au niveau du cortex cellulaire. Cet anneau est séquentiellement assemblé au cours de la mitose et se contacte à la fin de l'anaphase, au moment où le fuseau mitotique et désassemblé. La contraction, mais non pas l'assemblage, de l'anneau dépend d'un réseau de signalisation appelé septation initiation netvvork' ou SIN. Les composants centraux du SIN sont trois kinases (cdc7, sidi et sid2) ainsi que leurs sous-unités régulatrices (spgl, cdc14 et mob1, respectivement). La signalisation dépend du nucléotide rattaché à la GTPase spgl qui est régulée par une GAP comprenant deux sous-unités cdc16 et byr4. La signalisation est présumée provenir du pôle du fuseau où les composants centraux du SIN sont ancrés grâce à un échafaudage comprenant sid4 et cdcl 1. La signalisation est étroitement régulée pour assurer une bonne coordination entre mitose et cytokinèse. Une activation ectopique du SIN en interphase peut découpler la formation du septum de la mitose, engendrant des cellules à multiples septa qui ne sont pas clivés. C'est pourquoi les régulateurs du SIN sont d'un intérêt considérable. Cette étude se concentre autour de deux ces régulateurs, dma1 et ubc8. J'ai montré que dma1 devient essentiel quand la signalisation du SIN est activée. Ceci m'amène à proposer un modèle en trois parties pour la régulation du SIN durant la mitose. Une expression élevée de dma1 inhibe la signalisation du SIN et empêche la division cellulaire. Afin d'identifier des substrats ou médiateurs potentiels de la toxicité de dma1, des supresseurs en copies multiples ont été identifiés. Un de ces supresseurs, ubc8, constitue le deuxième sujet de cette thèse. Les études génétiques et moléculaires suggèrent un rôle inhibiteur du SIN par ubc8. Ubc8p est une protéine nucléaire, non essentielle, mais en son absence les cellules ne peuvent pas restreindre la fomation du septum, lorsque la progression de la mitose est perturbée. Les données suggèrent que ubc8 pourrait être un effecteur de point de contrôle de l'assemblage du fuseau mitotique. Prises dans leur ensemble, ces données apportent un nouvel éclairage sur les mécanismes de régulation de la cytokinèse dans S. pombe.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

The role of transcription factories-mediated interchromosomal contacts in the organization of nuclear architecture.

Using numerical simulations, we investigate the underlying physical effects responsible for the overall organization of chromosomal territories in interphase nuclei. In particular, we address the following three questions: (i) why are chromosomal territories with relatively high transcriptional activity on average, closer to the centre of cell's nucleus than those with the lower activity? (ii) Why are actively transcribed genes usually located at the periphery of their chromosomal territories? (iii) Why are pair-wise contacts between active and inactive genes less frequent than those involving only active or only inactive genes? We show that transcription factories-mediated contacts between active genes belonging to different chromosomal territories are instrumental for all these features of nuclear organization to emerge spontaneously due to entropic effects arising when chromatin fibres are highly crowded.

Effects of supercoiling on enhancer-promoter contacts.

Using Brownian dynamics simulations, we investigate here one of possible roles of supercoiling within topological domains constituting interphase chromosomes of higher eukaryotes. We analysed how supercoiling affects the interaction between enhancers and promoters that are located in the same or in neighbouring topological domains. We show here that enhancer-promoter affinity and supercoiling act synergistically in increasing the fraction of time during which enhancer and promoter stay in contact. This stabilizing effect of supercoiling only acts on enhancers and promoters located in the same topological domain. We propose that the primary role of recently observed supercoiling of topological domains in interphase chromosomes of higher eukaryotes is to assure that enhancers contact almost exclusively their cognate promoters located in the same topological domain and avoid contacts with very similar promoters but located in neighbouring topological domains.