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PubMed1994

Interphase cytogenetics of glioblastoma and gliosarcoma.

Interphase cytogenetics, i.e., in situ hybridization using probes to chromosome-specific DNA, enables histological identification of cells bearing numerical chromosome aberrations and cytogenetic analysis of composite tumors. We studied routinely processed tissues from seven glioblastomas and three gliosarcomas using biotinylated probes to pericentromeric alpha-satellite sequences on chromosomes 10, 17 and X. By applying various pretreatment protocols, an evaluable compromise between morphology and signal intensity was obtained in most cases. Compared to vascular cells with normal chromosomal counts, a significant subpopulation of glioblastoma cells showed monosomy 10 (four of five cases), monosomy 17 (one of seven cases) and loss of one X chromosome (one of seven cases). All monosomy 10 cases comprised additional areas where two copies of chromosome 10 were retained. Among the gliosarcomas, both the glioma and the sarcoma portion showed monosomy 10 in one case and monosomy 17 in another case. In contrast, in the third case of gliosarcoma, monosomy 10 was found only in the glioma portion, whereas a gain of chromosome X was observed in the sarcoma portion. We conclude that: (1) numerical chromosome aberrations can be detected in routinely processed brain tumor biopsy specimens using interphase cytogenetics, making retrospective studies feasible; (2) glioblastomas show intratumoral cytogenetic heterogeneity with formation of monoclonal cell clusters; and (3) sarcoma and glioma elements in gliosarcomas may exhibit the same or different numerical chromosome aberrations, suggesting various histogenetic pathways of the sarcoma-like portion.

Adult↗
PubMed1995

Immunostaining and interphase arrangement of field bean kinetochores.

More than 100 sera from patients with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were tested in order to detect antigenic nuclear components of the field bean Vicia faba (2n = 12). Kinetochores of mitotic chromosomes and prekinetochores of interphase cells from root-tip meristems were specifically labelled via an indirect immunofluorescence procedure by antibodies of one of these sera. In 44% of interphase nuclei in which centromeres could be identified, only half (6) of the number of expected prekinetochores (12) was detected, circumstantially indicating at least transient association of homologous centromeres. Some nuclei showed clustering of centromeres at one pole (Rabl configuration). In metaphase chromosomes, each sister kinetochore contained a fluorescent spot. Western blotting of field bean nuclear proteins revealed four antigenic proteins of 28, 30, 64 and 68 kDa.

Blotting, Western↗
PubMed1988

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes.

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

Biotin↗
PubMed1996

Painting rye B chromosomes in wheat: interphase chromatin organization, nuclear disposition and association in plants with two, three or four Bs.

The B chromosomes (Bs) of rye (Secale cereale) have been studied at interphase in terms of their chromatin organization, patterns of nuclear disposition and physical association in plants with two, three, and four Bs. The study was made in the Lindström strain of hexaploid wheat, which carries the rye Bs as an addition line, by in situ hybridization with a B-specific probe and by genomic in situ hybridization (GISH) with rye genomic DNA, enabling whole chromosome painting. Repetitive sequences common to the As and Bs of rye allow for visualization of the rye B at interphase in the wheat background. A B-specific probe enables the orientation of two or more Bs to be determined, and the combination of both probes used together gives information on the disposition of the Bs and on their patterns of physical association within the nucleus. The Bs form linear "strings', and the ends of their long arms, which can be detected by the B-specific probe, are usually located within the hemisphere of the nucleus that has the least condensed chromatin. There is dose-dependent association, and even numbers (2B, 4B) have a greater preference for association than odd ones (3B).

Chromatin↗
PubMed1978

Number of nucleoli in Ehrlich tumor cells during interphase.

Fractions of mouse Ehrlich ascites tumor cell populations with a high percentage of cell either in early or in late interphase were separated by centrifugation on ficoll gradients. Nucleoli were studied by light or electron microscopy in these cell subpopulations. It was shown that, in these cells, the number of nucleoli per nucleus does not vary significantly during interphase. This result is discussed and an anlysis of the relationships between the number and the volume of the nucleoli in these cells is present.

Animals↗
PubMed1982

The number of nucleolar silver-stained granules of active nucleolus organizer regions in mitotic and interphase cells of rat Yoshida and Zajdela ascitic tumors.

To provide more information on the number of silver-stained granules (SSGs) of active mitotic nucleolus organizer regions (NORs) as well as interphase nucleoli, rat Yoshida ascitic sarcoma and Zajdela ascitic hepatoma cells were studied by means of standardized one-step and two-step silver-staining procedures. The number of SSGs of mitotic active NORs was relatively constant and corresponded to the number of active NORs of animals bearing the tumors investigated. Some anaphases and telophases were "asymmetric", i.e. chromosomal figures (future nuclei) in one and the same cell contained different number of SSGs (active NORs). The incidence of such asymetric anaphases and telophases was higher in aneuploid (hypoploid) Zajdela hepatoma than in euploid (diploid) Yoshida sarcoma cells. In the interphase, the number of SSGs was low in small or large cells with distinct chromocenters or chromosomes condensation presumably representing postmitotic and premitotic cells. In contrast, the highest number of SSGs was noted in nucleoli of large cells which were usually characterized by a fine chromatin structure.

Aneuploidy↗
PubMed2000

The value of interphase fluorescence in situ hybridization for the detection of translocation t(12;21) in childhood acute lymphoblastic leukemia.

Translocation t(12;21)(p13;q22) is the most frequent cytogenetic abnormality in childhood acute lymphoblastic leukemia (ALL) and is generally associated with favorable prognosis. In this report, we assessed the value of dual-color interphase fluorescence in situ hybridization (FISH) for the detection of t(12;21). Fifty-three patients were screened for ETV6/CBFA2 fusion by means of FISH, using two cosmid probes mapped on ETV6 and on CBFA2, respectively. The cut-off value (mean + three standard deviations) for positivity established on control patients was 9.3%. A comparison between FISH and molecular methods [reverse-transcriptase polymerase chain reaction/Southern blot (RT-PCR/SB)] was possible in 52 patients: 34 of 52 (65.4%) showed negative results with both approaches, and 13 of 52 (25%) were positive; 5 of 52 (9.6%) showed discrepancies: four patients who were positive using RT-PCR/SB were negative using FISH. Conversely, one patient negative when using RT-PCR/SB was positive with FISH. Further investigations on this patients, cytogenetically characterized by add(12p), showed an atypical breakpoint on ETV6, located 5' to the common breakpoint. Compared with RT-PCR and SB, dual-color interphase FISH with the cosmid probe set proved to be highly specific but showed limited sensitivity.

Blotting, Southern↗
PubMed2002

Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies.

The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "nondenaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The nondenaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in nondenatured nuclei, if present. Probes such as (GA/TC)(n) and (GAA/TTC)(n) sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner.

Cell Line↗
PubMed2001

Cytogenetic abnormalities of alveolar soft-part sarcomas using interphase fluorescent in situ hybridization: trisomy for chromosome 7 and monosomy for chromosomes 8 and 18 seem to be characteristic of the tumor.

Four alveolar soft-part sarcomas were investigated by means of standard immunohistochemistry and interphase cytogenetics to further characterize the immunophenotype and proliferative activity of this tumor. The main goal of this study was to explore the chromosomal changes of this rare soft-tissue sarcoma. One epithelial (KLI), three neurogenic [neuron specific enolase (NSE), PGP 9.5, and S100], and five myogenic (desmin, myoglobin, alpha-smooth mnuscle actin, alpha-sarcomeric actin, and MyoD1) markers were used for the immunophenotypical analysis. Proliferative activity was assessed using the Ki67 index. Twelve (peri)centromeric (1, 3, 4, 6, 7, 8, 10, 12, 15, 17, 18, and X) and one telomeric (17q25-qtel.) chromosomal probes were used for interphase cytogenetic analysis. Three of the cases showed cytoplasmic desmin and/or myoglobin, and one showed smooth muscle actin positivity. All of the four tumors had granular, cytoplasmic, possibly nonspecific MyoD1 and sarcomeric actin positivity. Two of the tumors were positive for vimentin, four gave focal and weak staining with neurogenic markers (four of four NSE, one of four S100, and four of four PGP 9.5), but none of them was positive with KLI. Alveolar soft-part sarcomas may show myogenic immunophenotype in a number of cases, which supports myogenic differentiation. Fluorescent in situ hybridization using alpha satellite chromosomal probes revealed significant alterations in all of the cases. Most frequent and repeated numerical changes, which seem to be characteristic of the neoplasm and may play an important part in its pathogenesis and/or progression, were trisomy 7, monosomy 8 and monosomy 18.

Adolescent↗
PubMed2005

Avian genomes: different karyotypes but a similar distribution of the GC-richest chromosome regions at interphase.

The chicken karyotype, like that of the vast majority of avian species, shows a large number of dot-shaped microchromosomes that are characterized, like most telomeric regions of the macrochromosomes, by the highest GC levels and the highest gene densities. In interphase nuclei, these gene-dense regions are centrally located, and are characterized by an open chromatin structure (a similar situation also exists in mammals). Avian species belonging to the Accipitridae family (diurnal raptors) show a karyotype with no very large chromosomes, and with only a very small number of microchromosomes. To identify the GC-rich (and gene-rich) regions of the chromosomes and nuclei from Accipitridae, we performed heterologous in-situ hybridizations using chicken GC-richest isochores as probes. Our results clearly show that the gene-rich regions are prevalently located in the few microchromosome pairs and in the telomeric regions of the middle-sized chromosomes, as well as in the interior of the interphase nuclei. This result is consistent with a common organization of the genome in the nuclei of warm-blooded vertebrates. Indeed, in spite of the different size and morphology of the chromosomes, the gene-dense regions are always located in the interior of the nuclei.

Animals↗
PubMed1986

Direct evidence for the non-random localization of mammalian chromosomes in the interphase nucleus.

Indirect immunofluorescence staining with human anti-centromere autoantibodies from a patient (LU 851) suffering from the CREST form of scleroderma was used to analyse chromosome topology in interphase nuclei of rat-kangaroo (PTO) and Indian muntjac (IM) cells. In some cells, centromeres were arranged in pairs suggesting association of homologous chromosomes. Clustering of centromeres at one pole of the nucleus (Rabl configuration) and other patterns suggesting higher order organization were also observed. In one fifth of the IM cells it was possible to identify the intranuclear location of each single chromosome on the basis of the morphology of the immunostained centromeres. In 30% of the IM cells in which centromeres could be identified, homologous chromosomes occupied adjacent territories within the interphase chromatin.

Animals↗
PubMed1990

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells.

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.

Amniotic Fluid↗
PubMed1991

Dynamics of nucleolar fusion in neuronal interphase nuclei in vitro: association with nuclear rotation.

Nuclear rotation (NR) refers to the motion of chromatin domains in interphase nuclei of several cell types, including neurons, in vitro. It has been proposed that NR may function, during cellular differentiation, in the transposition of specific chromatin domains into the cytotypic chromosome pattern known to exist in interphase nuclei. It is controversial whether NR represents motion of nuclei in toto, including the nuclear envelope, or whether NR represents independent motion of subnuclear structures, relative to each other. Using nucleoli as markers of chromatin motion in dorsal root ganglion neurons in vitro, we now show that trajectories of individual nucleoli are spatially restricted to subnuclear domains. Nucleoli move at mean rates of 2.153 +/- 0.037 deg/min and exhibit periodic fluctuations in rate. Fast Fourier transform analyses show dominant frequencies ranging from 0.47 c/h to 2.91 c/h. The power spectra of periodic motion of 15 of 25 nucleoli monitored exhibit resonance which suggests that NR represents forced harmonic motion. Quantification of motion of nucleoli in differentiating, multinucleolate neurons showed that internucleolar distances may rapidly decrease, culminating in nucleolar fusion, and showed that nucleolar fusion was invariably associated with a transient increase in the rate of NR. These results indicate that nucleoli may move independently; that an association exists between rearrangement of chromatin domains and NR; and that NR, nucleolar fusion, and differentiation are linked.

Animals↗
PubMed1991

Arrangement of kinetochore proteins and satellite DNA in neuronal interphase nuclei: changes induced by gamma-aminobutyric acid (GABA).

Discrete chromatin domains occupy specific nuclear compartments in neuronal interphase nuclei. Nuclear rotation, defined as the motion of interphase chromatin domains, has been proposed to function in the placement of specific chromatin domains to nuclear compartments which are appropriate to the physiological state or the state of differentiation of the cell. Rates of this chromatin motion may be increased by agents, including gamma-amino butyric acid (GABA), which may cause changes in gene expression. To test whether GABA also causes a spatial rearrangement of specific chromatin domains, the three-dimensional distribution of kinetochores in nuclei of mouse dorsal root ganglion neurons was determined by immunofluorescence. In addition, centromeric satellite DNA sequences were localized by in situ hybridization using a biotinylated mouse satellite DNA probe followed by immunofluorescence. Kinetochores occurred in clusters, associated with nucleoli or in intermediate nucleoplasmic regions, between the nucleolus and the nuclear membrane. Clusters of satellite DNA sequences were found either associated with nucleoli or throughout the nucleoplasm. Strikingly, nucleoplasmic kinetochores consistently occupied a 5-microns distance from the nuclear center, representing 70% of the spherical nuclear radius. Exposure of neurons to GABA induced a significant reorganization of kinetochores which may represent movement of chromosomes to alternate nuclear compartments to accommodate a new transcriptional state.

Animals↗
PubMed1992

Protein phosphorylation is involved in the regulation of chromatin condensation during interphase.

Loci affecting the condensation state of interphase chromatin have been previously identified from analysis of suppression and enhancement of position effect variegation (PEV) in Drosophila. Here we show that Su-var(3)6 and an allelic mutant, e078, which both show suppression of PEV in the heterozygous state, have point mutations (Gly220-->Ser and Gly220-->Asp, respectively) in a protein phosphatase 1 catalytic subunit located at 87B (PP1 87B). The mutated glycine is conserved in all known protein serine/threonine phosphatases in the same gene family, and its substitution decreases PP1 activity. We conclude that protein dephosphorylation by PP1 87B regulates the condensation state of chromatin during interphase.

Amino Acid Sequence↗
PubMed1991

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis.

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

Autoantibodies↗
PubMed1994

Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes.

Increased frequencies of structural and numerical chromosomal aberrations have been observed in the lymphocytes of benzene-exposed workers. Similar aberrations occurring in bone-marrow cells may contribute to the increased incidence of leukemia seen in these populations. Fluorescence in situ hybridization with chromosome-specific DNA probes is a relatively new technique which shows promise for the identification of aneuploidy-inducing agents. In these studies, fluorescence in situ hybridization with several chromosome-specific DNA probes was used to investigate the ability of the benzene metabolite hydroquinone to induce hyperdiploidy in interphase human lymphocytes. Using a classical satellite probe specific for human chromosome 9, a significant dose-related increase in the frequency of cells containing 3 or more hybridization regions was observed following the in vitro exposure of lymphocytes to hydroquinone at concentrations from 75 to 150 microM. At the 100-microM concentration of hydroquinone, the frequency of nuclei containing 3 or more hybridization regions was determined using probes for chromosomes 1, 7 and 9. Significantly higher frequencies of affected nuclei were observed using the chromosome 1 and 9 probes when compared to the chromosome 7 probe. To establish whether this difference was due to the nonrandom involvement of these chromosomes in hydroquinone-induced hyperdiploidy or to chromosomal breakage within the chromosomal region targeted by these probes, a multicolor fluorescence in situ hybridization approach was developed using probes to two adjacent regions on chromosome 1. Using this tandem-labeling approach, the frequency of nuclei with multiple hybridization regions and the origin of the regions was determined by scoring slides labeled simultaneously with the chromosome 7 alpha satellite probe and the adjacent alpha and classical satellite probes for chromosome 1. The results of these studies confirmed that hydroquinone exposure resulted in a significant increase in hyperdiploid nuclei, but indicated that the different frequency of nuclei containing 3 or more hybridization regions observed using the chromosome 1 and 7 probes, was due to breakage within the chromosomal region targeted by the chromosome 1 classical satellite probe. These results indicate that hydroquinone may contribute significantly to the numerical and structural aberrations observed in benzene-exposed workers. In addition, the multicolor fluorescence in situ hybridization approach utilized in these studies promises to be a powerful technique for the detection of chromosomal breakage occurring in interphase human cells.

Benzene↗
PubMed1984

Comparison of metaphase and interphase nucleolar activity in Hela-CCL2 cells and PHA-stimulated human lymphocytes.

Metaphase and interphase nucleolar activity in cultured Hela-CCL2 cells and PHA-stimulated human lymphocytes have been studied with silver nitrate staining. In metaphase, we examined the relationship between the actual number of active NORs (AgNORs) and total number of NOR-bearing acrocentrics. Interphase silver staining over the nucleus was analyzed cytodensitometrically and morphologically. From all investigations, Hela-CCL2 cells and lymphocytes were shown to have similar levels of nucleolar activity. Our results suggest that there is a form of regulation of nucleolar activity in malignant Hela-CCL2 cells as compared to PHA-stimulated human lymphocytes.

Cell Nucleolus↗