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At least 307 records · Page 17Linked to original sources
PubMed2003

[Influence of irradiation on the dynamic three-dimension distribution of abl and bcr genes in the interphase nuclei of IM-9 cell].

OBJECTIVE: To investigate the material foundation of the fusion of bcr and abl genes, and to explore the pathogenesis of chronic myeloid leukemia. METHODS: By FISH combined with laser confocal scanning microscopy, the three-dimension (3D) distribution of bcr and abl genes in the interphase nuclei of normal and irradiated IM-9 cells was studied in each cell cycle phases. RESULTS: abl and bcr genes distributed non-randomly in the interphase nuclei of IM-9 cells. abl gene preferably located at the outer layer and bcr near the core of the nucleus. The two genes were drawn near each other most in G(0) phase. The relative distance between the homologous genes was greater at proliferation phase than at quiescence phase. After irradiation, the relative distances from the two genes to the core and between the two genes were shortened, with the shortest distance between the two genes in S phase. CONCLUSION: Irradiation could change the 3D-distribution of abl and bcr genes in the interphase nuclei of IM-9 cell and accelerate them to draw near each other.

Cell Nucleus↗
PubMed2003

[Detection of 20q(-) chromosome abnormality in myelodysplastic syndrome by interphase fluorescence in situ hybridization].

In order to explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of partial deletion of the long arm of chromosome 20 (20q(-)) in patients with myelodysplastic syndrome (MDS), spectrum Green fluorescein directly labeled yeast artificial chromosome (YAC) clone 912C3 which spans the breakpoint cluster region in band 20q12 was used as probes to perform interphase FISH on the marrow cells from 52 cases of MDS and 5 normal controls. 200 to 300 cells were scored for each case and cases which had cells with a green hybridization signal>7.16% were defined as 20q(-) positive. The results of FISH were compared with those of conventional cytogenetics (CC) assay. The results showed that among 52 cases of MDS, 7 (13.5%) cases were positive by FISH, however, of which, 4 cases were positive and the other 3 cases were negative by CC assay. It is concluded that YAC912C3 and interphase FISH providing a powerful technique in the detection of 20q(-) in MDS is an important complement to CC assay.

Adolescent↗
PubMed2004

[Analysis of the frequency of spontaneous aneuploidy in human somatic cells using interphase cytogenetic technology].

The frequency of spontaneous aneuploidy of the four autosomes and sex chromosomes in the interphase nuclei of cultivated and uncultured lymphocytes from clinically healthy men was examined by use of two-color fluorescent in situ hybridization (FISH). It was shown that in uncultured cells from the individuals examined autosomal nullosomies were practically not detected (the frequency 0 to 0.01%). At the same time, the frequency of such cells with either Y, or X nullosomy was at least an order of magnitude higher (about 0.15%). This frequency was comparable with the level of Y- or X-disomic cells, and also with autosomal monosomies, precluding from considering the X-nullosomic cells as hybridization artifacts. During lymphocyte cultivation, a statistically significant increase in the total frequency of Y- or X-nullosomic cells was observed already after the first cell division cycle. Thus, interphase FISH analysis is a sufficiently sensitive method enabling detection of higher, compared to the autosomes, loss of sex chromosomes in the process of cell division, a phenomenon observed during replicative cell aging, as well as during natural aging of the organism. Male cells with the de novo lost singular X chromosome, probably, switch to apoptosis and do not survive during further life of a cell population. The frequency of total aneuploidy in human somatic cells with the correction for the resolution capacity of the interphase FISH analysis constituted 5.62 and 6.90% for uncultured and cultivated cells, respectively. This aneuploidy level is close to that in spermatozoa. The data obtained can serve as the basis for the examination of the aneugenic (aneuploidy-inducing) genotoxic effects and for the analysis of interindividual genetic instability.

Aneuploidy↗
PubMed1992

[Interphase cytogenetic studies of human X chromosome].

The chromosome in situ hybridization with human X chromosome alpha satellite DNA probe (pBamX7) on human lymphocyte metaphases and interphase nuclei was performed for interphase cytogenetic studies. The individuals with numerical or structural abnormalities of X chromosome were studied. The results showed that the probe hybridized specifically to the centromeric region (p11----q11) of X chromosome. The number of silver grain clusters in interphase nuclei was correlated with that of X chromosome. Most of the clusters located near the nuclear membrane where inactive X chromatins (Barr-bodies) were usually found. The method of ascertaining the number of X chromosomes by in situ hybridization was much more reliable than that by counting the number of Barr-bodies. The modified R-banding technique was introduced and the significance of this work was also discussed.

Chromosome Aberrations↗
PubMed1992

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.

Adult↗
PubMed2005

[The application of interphase fluorescence in situ hybridization to the diagnosis of X chromosomal count abnormality in ovarian carcinoma cell].

To study the technique of fluorescence in situ hybridization (FISH) and its application value in the diagnosis of sex chromosomal count abnormality in ovarian carcinoma cell. Biotin labeled alpha satellite X chromosome DNA(pBamX7) probe was hybridized with pre-treated slides of ovarian carcinoma cell interphase nucleus in 18 cases of ovarian carcinoma specimens. The slides were treated with Avidin-FITC and Anti-avidin, amplified with an additional layer and counter-stained with PI in antifade solution. The hybridization signals as well as interphase nucleus settings were observed with WIB filters under fluorescence microscope Olympus AX-70, and the number of interphase nucleus in the ovarian carcinoma cell was counted. It was observed under the microscope that the Biotin labeled pBamX7 probe showed green hybridization signals. Cytoplasm counter-stained with PI showed reddish orange. Increased chrosome X copy number was observed in 11/18(61%) ovarian carcinoma specimens, of which the rest 7 (39%) had no increase of chrosome X copy number. Gain of X chrosome had a certain incidence in ovarian cancers, which played a role in the recurrence and development of ovarian cancers. Its significance needs further investigation.

Adult↗
PubMed1991

Trisomy 12 in chronic lymphocytic leukemia: an interphase cytogenetic study.

Interphase cytogenetics by means of in situ hybridization with the chromosome 12-specific biotinylated alpha satellite DNA probe pSP 12-1 was used for the study of trisomy 12, the most common chromosomal abnormality in chronic lymphocytic leukemia. In situ hybridization was performed on methanol/acetic acid fixed cells of conventional cytogenetic preparations from eight patients and on morphologically and immunologically classified cells of cytospin preparations from seven patients. The results show that trisomy 12 is more common than assumed on the basis of karyotype analysis of metaphase chromosomes: 2 of 13 patients with a normal karyotype in G-banding analysis were shown to have trisomy 12 by interphase cytogenetics. Immunophenotyping of the cells of one patient showed that the trisomy was restricted to cells with Ig light chain clonality. For the evaluation of the prognostic, therapeutic, and biologic significance of trisomy 12, in situ hybridization should be used in parallel with karyotype analysis because it allows the study of all cell populations of both interphase and mitotic cells, whether neoplastic or normal.

Aged↗
PubMed1991

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.

Aneuploidy↗
PubMed1989

Effects of hyperthermia on chromatin condensation and nucleoli disintegration as visualized by induction of premature chromosome condensation in interphase mammalian cells.

The effects of hyperthermia on chromatin condensation and nucleoli disintegration, as visualized by induction of premature chromosome condensation in interphase mammalian cells, was studied in exponentially growing and plateau phase Chinese hamster ovary cells. Exposure to heat reduced the ability of interphase chromatin to condense and the ability of the nucleolar organizing region to disintegrate under the influence of factors provided by mitotic cells when fused to interphase cells. Based on these effects treated cells were classified in three categories. Category 1 contained cells able to condense their chromatin and disintegrate the nucleolar organizing region. Category 2 contained cells able to only partly condense their chromatin and unable to disintegrate the nucleolar organizing region. Category 3 contained cells unable to condense their chromatin and unable to disintegrate the nucleolar organizing region. The fraction of cells with nondisintegrated nucleoli increased with increasing exposure time at 45.5 degrees C and reached a plateau at almost 100% after about 20 min. Exponentially growing and plateau phase cells showed similar response. Recovery from the effects of heat on chromatin condensation and disintegration of the nucleolar organizing region depended upon the duration of the heat treatment. For exposures up to 15 min at 45.5 degrees C, a gradual reduction in the fraction of cells with nondisintegrated nucleoli was observed when cells were allowed for repair at 37 degrees C. However, only a very limited amount of repair was observed after a 30-min exposure to 45.5 degrees C. The repair times observed at the chromosome level were similar to those reported for the removal of excess protein accumulating in chromatin or the nuclear matrix, suggesting a causal relationship between the two phenomena. It is proposed that nuclear protein accumulation on chromatin or in the nuclear matrix reduces the accessibility of chromatin to enzymes responsible for the phosphorylation reactions necessary for chromatin condensation and disintegration of the nucleolus.

Animals↗
PubMed1988

Changes in a nuclear matrix antigen during the cell cycle: interphase and mitotic cells.

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin-stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two-dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.

Antibodies, Monoclonal↗
PubMed1987

Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study.

The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.

Animals↗
PubMed1995

Translocations and amplification of the BCL2 gene are detected in interphase nuclei of non-Hodgkin's lymphoma by in situ hybridization with yeast artificial chromosome clones.

Translocation of the BCL2 gene in B-cell malignancies carrying t(14;18) and amplification of the BCL2 gene in a cell line (HBL-2) derived from a non-Hodgkin's lymphoma (NHL) were detected specifically in both metaphase spreads and interphase nuclei by fluorescence in situ hybridization (FISH) using yeast artificial chromosomes (YACs). A YAC clone containing the BCL2 gene yA153A6, a 360-kb clone spanning from approximately 60 kb upstream of BCL2 exon 1 to approximately 60 kb 3' of the minor breakpoint cluster region, was used for single-color FISH analysis. Seven patients with NHL and one patient with acute lymphoblastic leukemia were analyzed for BCL2 translocations. Interphase nuclei of NHL patients showed three signals when hybridized with the yA153A6 probe. This was expected because the YAC clone spans the BCL2 breakpoint regions on 18q21.3. In a patient with acute lymphoblastic leukemia, a positive signal for BCL2 was detected on der(14) at band 14q32.33 by single-color FISH with the yA153A6 probe, whereas no signals were detected on der(18). The amplification of BCL2 in the HBL-2 cell line was observed on a characteristic abnormal chromosome 18, add(18)(q23); the periodic pattern of the fluorescent signal of this region was suggestive of an amplicon. Using double-color FISH with YAC clones containing the more centromeric 18q21.3 gene gastrin-releasing peptide (y302F10) and the 14q32.33 gene (IgH; Y6), we detected t(14;18) by showing the juxtaposition of the 18q21.3 and 14q32.33 bands on the derivative chromosome 18. Interphase FISH with these YAC clones provided a rapid procedure for the diagnosis of B-cell malignancies carrying t(14;18). In addition, we showed that translocations and amplification of the BCL2 gene can be detected at the single-cell level.

Adult↗
PubMed1995

Multiple myeloma: high incidence of chromosomal aneuploidy as detected by interphase fluorescence in situ hybridization.

Because metaphase cytogenetic studies in multiple myeloma (MM) are hampered by a low proliferative activity of myeloma cells in vitro, interphase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We therefore investigated chromosomal aneuploidy in 36 patients with MM using interphase FISH and alpha-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients (88.9%) were aneuploid for at least one of the chromosomes examined. In 24 patients (66%), aberrations of > or = 3 chromosomes were observed. Aneuploidy was predominantly characterized by a gain of chromosome numbers, with involvement of chromosomes 3, 7, and 11 occurring in > 50% of patients. Loss of a centromeric signal suggesting monosomy was most frequently observed for chromosomes 17 (22.2% of patients) and X (monosomic in 42.3% of female patients, but loss of chromosome X was never observed in males, P < 0.05). Dual-color FISH studies provided evidence for marked heterogeneity of aneuploid cells in 8 patients (22.8%). Occurrence of chromosomal aneuploidy was independent of stage and pretreatment status. Gain of chromosome 3 was significantly correlated with an IgA paraprotein (P < 0.05). In 12 patients, the direct comparison of metaphase cytogenetics and FISH showed that FISH detected aneuploidy of chromosomes in 9 patients that was missed by metaphase analysis. In conclusion, interphase FISH, by which chromosomal aneuploidy was detected in almost 90% of patients with MM, represents an approach for evaluating the clinical significance of specific chromosomal abnormalities in MM.

Adult↗
PubMed1995

Distribution of a Ca2+ storing site in PtK2 cells during interphase and mitosis. An immunocytochemical study using an antibody against calreticulin.

To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.

Animals↗
PubMed1995

Detection of CDKN2 deletions in tumor cell lines and primary glioma by interphase fluorescence in situ hybridization.

Deletions of chromosomal band 9p21 have been detected in various tumor types including melanoma, glioma, lung cancer, mesothelioma, and bladder cancer. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) has been proposed as a candidate tumor suppressor gene because it is frequently deleted in cell lines derived from multiple tumor types. We performed fluorescence in situ hybridization (FISH) with interphase cells using yeast artificial chromosome clones and a cosmid contig of the CDKN2 region. In 10 cell lines (4 glioma, 2 melanoma, 2 non-small cell lung cancer, 2 bladder cancer) with 9p alterations detected by molecular or cytogenetic analysis, interphase FISH with the CDKN2 cosmid contig detected all 9p deletions previously identified by molecular analysis. Using this probe, FISH analysis of primary glioblastoma tumors revealed homozygous deletions of the CDKN2 region in 6 of 9 tumors (67%) whereas a yeast artificial chromosome probe containing the interferon type I (IFN) gene cluster was deleted in only 4 cases (44%). Thus, it is likely that the CDKN2 region is the target of 9p deletions in gliomas. Interphase FISH will play an important role in defining the clinical significance of 9p deletions in primary tumors because it is especially applicable to clinical samples which may be contaminated by normal cells.

Brain Neoplasms↗
PubMed1994

Detection of aberrant clones in nearly all cases of angioimmunoblastic lymphadenopathy with dysproteinemia-type T-cell lymphoma by combined interphase and metaphase cytogenetics.

Trisomy 3, trisomy 5, and an X additional chromosome are the most frequent chromosome aberrations in angioimmunoblastic lymphadenopathy with proteinemia (AILD)-type T-cell lymphomas. To evaluate the frequency of +3 and +X clones, fluorescence in situ hybridization studies with centromere-specific probes for chromosome 3 and X were done in 41 patients with peripheral T-cell lymphomas (PTL). With this interphase cytogenetic approach, 32 of 41 patients (78%) showed +3 clones, and 14 patients (34%) +X clones. These frequencies far exceeded those observed with metaphase cytogenetics (+3, 41%; +X, 20%). Summing up the results of metaphase and interphase cytogenetics, aberrant clones were found in 37 of 41 patients with PTL (90%) and 32 of 36 patients with AILD-type T-cell lymphoma (89%). Although AILD-type T-cell lymphoma is considered a neoplastic disease, it is an exception in that it shows a high frequency of cytogenetically unrelated clones and single cells that cannot be derived from a common cell of origin because of their completely different karyotypes. In five patients, double hybridization with centromere-specific probes for chromosomes 3 and X showed that these aberrations occurred in different cells. When the results of metaphase and interphase cytogenetics were combined, 17 of 36 patients with AILD-type T-cell lymphoma (47%) had unrelated clones. This high frequency of oligoclonal proliferations may be caused by increased genetic instability and an immune defect resulting in impaired elimination of aberrant cells.

Blood Protein Disorders↗
PubMed1994

[Difference in the distribution and content of nucleolar protein B23 between interphase and mitotic HeLa cells].

Protein B23 is one of the two major protein components in eukaryocytic nucleolus. Previous studies indicate that the cellular content and distribution of protein B23 are correlated with the synthetic rate of ribosomal RNA (rRNA) and cell growth. It is more abundant in tumor and growing cells than in normal resting cells. It may translocate from nucleolus to nucleoplasm after serum deprivation, or after being treated with RNA inhibitors such as actinomycin D, toyocamycin, or doxorubicin. In order to search for the role of protein B23 in cell division, using an anti-B23 monoclonal antibody, we detect the cellular content and distribution of protein B23 in interphase and mitotic HeLa cells, respectively. Two microtubule drugs, colcemid and taxol, are used to arrest the mitotic cells. The results show the cellular content of protein B23 in mitotic cells is much higher than that in interphase cells. The intracellular distribution of protein B23 in mitotic cells is distinguished from that in interphase cells too.

Blotting, Western↗
PubMed1993

Intercellular interactions in the interphase death of irradiated thymocytes.

The effect of the interaction of different types of cells on the interphase death and pycnosis of thymocytes irradiated in vitro was studied. When removed from the thymus suspension of cells with natural killer activity, medullary thymocytes and macrophages did not change the radiation-induced death of cortical thymocytes. On the other hand, postirradiation incubation of cortical thymocytes together with unirradiated thymocytes or with cells of certain other cell lines diminished thymocyte death. Mixing the cell suspensions and changing the incubation medium decreased thymocyte death. All of these results indicate that these cells produce soluble mediators that are toxic to the cells that secrete them. The possible nature of these autotoxic mediators has been studied using inhibitors of arachidonic acid metabolism. Inhibitors of phospholipase A2 or lipoxygenase reduced interphase death markedly, while an inhibitor of cyclooxygenase did not. These data suggest that some lipoxygenase products may serve as autotoxic mediators in the interphase death of thymocytes.

Animals↗