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Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock.

Heat shock induces changes in G1 CHO cell nuclear matrix (NM) ultrastructure that may be related to heat-induced nuclear protein accumulation (Wachsberger and Coss, 1993, J. Cell. Physiol., 155:615-634). The present study quantitates recovery of alterations in NM fine structure in CHO cells heated in G1 and compares structural recovery with recovery of bulk RNA synthesis and surviving fraction (SF). Morphology of NM preparations was quantified 30 min and 20 hr following heat shock by 1) measurement of the number of fiber anastomosing points per unit area per NM, and 2) measurement of the length of fibers between points of anastomoses within individual NMs. Architectural recovery was nearly complete within 20 hr in cells heated at 43 degrees C or 45 degrees C with SFs of 0.27 or greater. No recovery of architecture was observed in heated cells with SFs of approximately 0.01 or less. The residual damage to NMs was associated with RNA-containing fiber networks as determined by means of RNase gold labeling. Recovery from inhibition of RNA synthesis following heat shock was related to recovery of NM architecture. It is suggested that 1) repair of NM architecture does not require full recovery of bulk RNA synthesis, and 2) partial or complete irreversible collapse of the NM may be responsible, in part, for heat-induced, interphase cell death.

Animals↗

Formation of flagella during interphase in secondary spermatocytes from Xenopus laevis in vitro.

In cell culture, single motile flagella, 1 micron in length, were observed to grow from secondary spermatocytes of Xenopus laevis within 2-3 hours after telophase I, at 22 degrees C. About 90% of the secondary spermatocytes formed flagella as observed by phase-contrast microscopy. The flagella grew up to 2-6 microns in length during interphase II, which lasted about 18 hours. The presence of the "9 + 2" microtubular structure of the flagellar axonemes of secondary spermatocytes was confirmed by electron microscopy. When chromosomal condensation began (prophase II), the flagella were resorbed into the cells and, after the second meiotic division, a flagellum was formed again by each of the round spermatids. Thus, there appears to be a close relationship between the meiotic division cycle and the formation of flagella. The possible contribution of Sertoli cells to the formation of flagella in secondary spermatocytes was examined by reducing the number of Sertoli cells to less than ten per culture. Under these conditions, flagella formed in secondary spermatocytes with very high efficiency. It is very likely that secondary spermatocytes form flagella in vivo, since the secondary spermatocytes were observed to have flagella immediately after dissociation of the testes.

Animals↗

AgNOR area in interphase nuclei of human tumours correlates with the proliferative activity evaluated by bromodeoxyuridine labelling and Ki-67 immunostaining.

The area of silver-stained proteins associated with interphase nucleolar organizer regions (AgNORs) was compared with labelling data obtained by bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining in 25 tumours of different origins and two non-neoplastic lesions of the thyroid. Our data demonstrate a highly significant correlation between the mean area occupied by the AgNOR proteins measured by an image processing system and the proliferative indices evaluated by BrdU labelling (r = 0.89, P less than 0.001) and Ki-67 immunostaining (r = 0.86, P less than 0.001). AgNOR protein area measurement is therefore proposed as a simple, inexpensive, and reliable method of evaluating the proliferative activity in routinely processed tumour samples.

Breast Neoplasms↗

Chorionic villus metaphase chromosomes and interphase nuclei analysed by chromosomal in situ suppression (CISS) hybridization.

Metaphase chromosomes and interphase nuclei of chorionic villus samples (CVS) in five cases were studied after treatment with trypsin and post-fixation in formaldehyde by chromosomal in situ suppression (CISS) hybridization. Our modified protocol enables the use of in situ hybridization techniques on CVS preparations after 42 h of culture. A balanced translocation and trisomy 13 were identified with the aid of CISS hybridization.

Chorionic Villi Sampling↗

Evaluation of X, Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization.

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.

Amniotic Fluid↗

The role of Ca ions in restoration of the structure of interphase and mitotic chromosomes in PK living cells after hypotonic stress.

The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK cells during their adaptation to hypotonic medium was studied. The recondensation process was found to be slowed down by the modification of plasma membrane with low concentrations of glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The effect of glutaraldehyde can be rapidly reversed by the addition of Ca(2+)-ionophore A23187. Intracellular Ca(2+)measurements show that the adaptation to hypotonic shock is accompanied by restoration of free Ca concentration, whereas the delay of chromatin condensation in glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the role of low concentration of Ca(2+)in chromatin compactization in vivo are discussed.

Animals↗

Formation of the male pronucleus, organization of the first interphase monaster, and establishment of a perinuclear plasm domain in the egg of the glossiphoniid leech Theromyzon rude.

Whole-mounted or sectioned eggs of the glossiphoniid leech Theromyzon rude were studied under the dissecting, fluorescence, light, and electron microscope. The egg is often penetrated by a single sperm that enters the animal hemisphere and becomes subjected to migration block. The latter is released shortly before or after discharge of the first pole cell, when the sperm centrosome initiates aster formation, the nucleus begins to be untwisted, and its chromatin decondensed. Sperm centration occurs along one side of the egg and appears to follow an arc-like trajectory as a result of vegetal and inward movements affected by colchicine and cytochalasin B but not by taxol. Results indicate that growing microtubules are needed for both movements, whereas actin filaments are essential for the vegetalward movement only. The sperm centrosome becomes the main microtubule organizing center (MTOC) of the egg and concomitantly originates the elaborate first interphase monaster. Additional peripherally situated MTOC form cytaster-like bodies whose visualization is improved by taxol treatment. A voluminous centrosphere, formed around the sperm centrosome, becomes a center of organelle accumulation, giving rise to a perinuclear plasm domain. This process seems to involve both import and replication of organelles.

Animals↗

Cell cycle-dependent distribution of telomeres, centromeres, and chromosome-specific subsatellite domains in the interphase nucleus of mouse lymphocytes.

Fluorescence in situ hybridization and 3-D image analysis were combined to study the distribution of specific chromosome subdomains through the course of the cell cycle in cultured mouse lymphocytes. DNA probes specific for major satellite DNA, minor satellite DNA, telomeric DNA and to chromosome X-, 8-, and 14-specific subsatellite DNA sequences were used. We demonstrate that a redistribution of the chromatin occurs during the cell cycle in the interphase nucleus, and that the profile of the rearrangement is highly dependent on the nature of the domain considered (centromeres, telomeres, or subsatellite regions). However, the relative arrangement of chromosome homologs to each other does not appear to be spatially defined or regulated.

Animals↗

A light microscope study of linker histone distribution in rat metaphase chromosomes and interphase nuclei.

Several subtypes of the linker histone H1 are present in normal rat kidney epithelial cells (NRK-52E). Although H1 is essential in nucleosome and chromatin packaging or condensation, the unique functions of these very basic proteins are largely unknown. There has been much speculation on the role of each H1 variant on developmentally regulated or tissue specific gene expression. We have examined the global distribution of several H1 subtypes on metaphase chromosomes in an attempt to uncover large-scale differences in chromatin condensation. Polyclonal antibodies raised against HPLC-purified rat H1 subtypes revealed a pattern much like G or Q bands for all H1 variants tested on chromosomes harvested with either aqueous or organic spreading methods. H1(0), a less abundant form of H1, may be associated with terminally differentiated or senescent cells. In cultures treated to induce higher levels of H1(0) there were no visible differences at the light microscope level in the antibody banding pattern between induced and noninduced cells. The distributions of H1 subtypes on chromosomes may be visible in different tissues when viewed at higher magnifications. While chromosome patterns were consistent with the antibodies tested, the interphase nuclei displayed clear differences. An epitope specific for anti-H1A antibody is present in the nuclear envelope and is possibly used for chromosomal location or anchorage. Anti-H1B antibody did not specifically label the nuclear envelope, nor did anti-H1(0) antibody. Highly concentrated regions of H1(0) surround the nucleoli, possibly indicating a cluster of genes that are poised for transcription.

Animals↗

Repositioning of human interphase chromosomes by nucleolar dynamics in the reverse transformation of HT1080 fibrosarcoma cells.

An experimental system which should be valuable for studying the role of spatial positioning of the nuclear genome in human cell function has been developed. Reverse transformation of the malignant HT1080 fibrosarcoma cell line upon treatment with 8-chloro-cAMP results in growth inhibition, cytoskeletal reorganization, changes in nuclear shape and chromatin accessibility, and formation of prominent nucleoli. Fluorescent in situ hybridization was used to study DNA positioning during nuclear remodeling. Morphometric analysis of the hybridization sites for both repetitive sequences and "painting probes" for whole chromosomes indicated dispersal of acrocentric chromosomes in untreated cells and a highly organized central location of these ribosome gene-containing chromosomes in association with one or a few large nucleoli in nondividing treated cells. The results suggest that there was a directed movement of interphase chromosomes during a response which normalized a malignant cell line. These large-scale repositionings may serve two functions in restoring a normal transcriptional setup to the nucleus. First, ribosome genes are placed in the nucleolus, their transcriptional suborganelle. Second, nucleolar anchorings together with additional perinucleolar centromeric associations orient the domain shapes of entire chromosomes, installing gene-rich chromosomal regions into pockets of (accessible) DNAse I-sensitive chromatin populated by spliceosomes.

8-Bromo Cyclic Adenosine Monophosphate↗

Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit.

The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.

Animals↗

Distribution and characteristics of betaII tubulin-enriched microtubules in interphase cells.

We have used a polyclonal antibody (Ab196) that specifically recognizes the betaII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual beta isotypes. Using photoimaging analysis, we observed betaII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of betaII-enriched microtubules together with the reported increased levels of betaII tubulin in taxol-resistant cells (M. Haber et al., 1995, J. Biol. Chem. 270, 31269-31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, betaII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-betaII tubulin antibody but not by anti-acetylated alpha tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.

Amino Acid Sequence↗

DNA global hypomethylation in EBV-transformed interphase nuclei.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.

Blotting, Southern↗

Type I protein kinase a is localized to interphase microtubules and strongly associated with the mitotic spindle.

We show here that type I protein kinase A is localized to microtubules during the entire cell cycle in epithelial (hepatoma, cervical carcinoma) and nonepithelial (myoblast) cell lines. The association of the type Ialpha regulatory subunit is very strong in all phases of mitosis, from prophase to cytokinesis. In interphase, the association appears weaker, reflecting perhaps a more dynamic molecular interaction. This regulatory subunit appears to recruit catalytic subunits as the latter are also associated with microtubules. BW1J hepatoma cells, stably transfected with either wild-type or mutant Ialpha regulatory subunit, are enriched in aberrant mitoses with multipolar spindles and in mono- or multinucleated giant cells. This suggests that type I protein kinase A could have a role in centrosome duplication and/or segregation, sister chromatid separation, or cytokinesis.

Animals↗

Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei.

The epidermal differentiation complex (EDC) at 1q21 is host to many structurally and functionally related genes coding for proteins involved in the differentiation process of keratinocytes. The grouping together of these genes which share spatial and temporal expression and interrelated functions is a remarkable genomic feature which has led to suggestions that the region may have a coordinated transcription control mechanism. With the growing awareness that the organization of the genome within the interphase nucleus is relevant to transcriptional activity, we have investigated the spatial organization of the EDC in the nuclei of keratinocytes, where the EDC genes are highly expressed, and lymphoblasts, where they are silent. Using 2D and 3D FISH we find that in keratinocyte nuclei the EDC is frequently positioned external to the chromosome 1 territory compared to lymphoblasts where the EDC more often adopts a peripheral or internal location. It has been previously shown that the MHC region can extend from the chromosome 6 territory in relation to transcriptional activity. This study of the EDC thus provides a further example of a gene-dense complex capable of assuming extraterritorial positioning in relation to cell type/transcription status.

B-Lymphocytes↗

c-myc and chromosome 8 centromere studies of ovarian cancer by interphase FISH.

Forty tumor specimens from patients with ovarian cancer were studied for amplification of the c-myc oncogene relative to chromosome 8 centromere number using dual-color FISH. Interphase cytogenetic analysis showed amplification of the c-myc oncogene in 40% (16/40) of tumors using the standard oncogene:centromere ratio method of analysis. Eleven of these showed moderate amplification of c-myc, and 5 samples showed high amplification. Eight of the sixteen (50%) amplified tumors were polysomic centromere 8 as were 14 of the 24 (58%) non-amplified tumors. In previously reported work with these samples, the oncogene HER-2/neu, the chromosome 17 centromere, and the tumor suppressor gene p53 had been studied. When using the standard oncogene:centromere ratio criteria, 5 samples had amplification of both the c-myc and the HER-2/neu oncogenes, 5 samples had HER-2/neu amplification but not c-myc, 11 samples had c-myc amplification but not HER-2/neu, and 19 samples had neither oncogene amplified. The p53 gene was found to be deleted in 22.5% (9/40) of samples. The loss of the p53 gene did not appear to have any clinical correlation. The presence of an extra centromere 8 also did not appear to have any clinical correlation. The Kaplan-Meier survival curve for those patients who have c-myc amplification, while not statistically significant, appears to show a trend toward poorer survival. The survival curve for patients whose tumors have HER-2/neu amplification shows no clinical significance. It is of great interest, however, that the Kaplan-Meier plot of survival for patients whose tumors have amplification of both c-myc and HER-2/neu shows a significant difference (P = 0.047). The median survival times of the doubly amplified patient group and the non-doubly amplified groups were 12 and 43 months, respectively. This is the first study of the oncogene c-myc using FISH. The results suggest that the amplification of c-myc may indicate a poorer patient survival and that the amplification of both c-myc and HER-2/neu in combination may be a better prognostic indicator of poor patient survival.

Adult↗

Interphase fluorescence in situ hybridization for trisomy 12 on archival ovarian sex cord-stromal tumors.

Trisomy 12 is a nonrandom chromosomal abnormality found in a large proportion of ovarian sex cord-stromal tumors (OSCTs), including thecoma-fibromas (TFs) and granulosa cell tumors (GCTs). The prognostic significance of trisomy 12 in these tumors, however, is unknown. A series of 16 OSCTs, obtained from patients with long-term follow-up, was analyzed for the presence of trisomy 12 by interphase fluorescence in situ hybridization on paraffin-embedded sections. Sections of the contralateral nonneoplastic ovary were available in five cases and utilized as controls. Evidence of trisomy 12 was detected in 9 of 10 TFs, and contrary to previous reports, in only one of six GCTs. One TF with trisomy 12 was a malignant variant that resulted in the death of the patient in 5 months, but the remaining TFs with trisomy 12 were cytologically and clinically benign in those with follow-up available. The single GCT with trisomy 12 was a nonaggressive, stage 1 lesion without evidence of recurrence after 264 months, whereas those GCTs without trisomy 12 included one stage 2 tumor and a cytologically atypical GCT with tumor necrosis and an elevated number of mitotic figures. The evidence suggests that the great majority of OSCTs with trisomy 12 is clinically benign, but not all benign OSCTs have trisomy 12. We conclude that the presence of trisomy 12 is of limited prognostic usefulness in OSCTs.

Adult↗

Interphase fluorescence in situ hybridization studies of ovarian adenocarcinomas using the midisatellite probe.

The genes involved in ovarian carcinogenesis are largely unknown. Cytogenetic studies have shown a large number of chromosomal abnormalities in ovarian cancers. Molecular studies have additionally found abnormalities. Few in situ hybridization studies have been performed on ovarian cancer tissues. We chose to study the distal region of chromosome 1p with the midisatellite probe and interphase fluorescence in situ hybridization. A total of 35 patient samples, including various controls and cancers, was collected from our pathology archives. Our cancer cases included some patients with stage I disease, in whom tumors arose in endometriotic cysts. In these cases, both tumor tissue and areas in the cyst distant from the tumor mass were examined. Results showed clear cell carcinoma nuclei to have an increase in both number and size of probe signals, interpreted as representing amplification of the probed region of chromosome 1. Serous carcinomas showed an increase in the number of signals, up to four. We felt this could be a result of amplification, or, because these cells exhibited the highest mitotic counts, to DNA doubling in preparation for mitosis. Endometrioid carcinomas resembled controls in showing up to two small probe signals, but not more. We conclude that amplification in distal chromosome 1p occurs in ovarian clear cell, and possibly serous, carcinomas and may not be important in endometrioid carcinomas. Because alteration was not found in the various control epithelia, including nonmalignant-looking areas from cysts which also contained cancer, we believe that the change, when present, may not be an early step in carcinogenesis.

Adenocarcinoma↗