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PubMed1990

The mechanism of radiation-induced interphase death of lymphoid cells: a new hypothesis.

The interphase death of irradiated rat thymocytes depends on their concentration during postirradiation incubation. The kinetics of pycnosis and cell death determined with the trypan blue exclusion test in the samples with the highest cell concentration (1-2 x 10(7) cells/ml) is consistent with the data available in the literature, whereas the samples with the lowest concentration (2 x 10(5) cells/ml) undergo almost no pycnosis and death after irradiation with doses up to 50 Gy. On the basis of these results, we suggest a new mechanism of interphase death involving an interaction between irradiated thymocytes and the fraction of thymus cells possessing cytocidal activity. The observed correlation between the cytocidal activity and interphase death of thymocytes from animals of different ages favors our mechanism. It was found that the inhibitors which prevent the conjugation of killer cells and their targets do not influence interphase death, while the substances which block the secretion of cytotoxic factors or their action on the target membrane do protect from interphase death. Thus we suggest that the irradiation activates the killer cells to secrete some cytotoxic factors which induce pycnosis and interphase death of thymocytes.

Animals↗

PubMed1979

Visualization of the interphase chromosomes of Ornithogalum virens and Muntiacus muntjak.

A technique for visualizing "interphase chromosomes" was applied to nuclei of the angio-spermous plant, Ornithogalum virens (2 n = 6), and the male mammal, Muntiacus munjak (2 n = 7), in an attempt to correlate the numbers of "chromosomes" visible during interphase with the respective diploid chromosome numbers. The alterations in chromosome structure observed during G1, S, and G2 periods were comparable to those previously reported in Allium cepa and Chinese hamster (CHO line) cells [33], but for technical reasons it was only possible to make accurate counts of interphase chromosomes in the G1 nuclei of O. virens. In addition, from our observations of interphase chromosomes that were pulse-labelled with tritiated thymidine and a parallel study of premature chromosome condensation (PCC) using pulse-labelled M.muntjak cells, we conclude that, although chromatin decondensation may be required for DNA synthesis, extreme chromatin decondensation can occur in the absence of DNA synthesis. Generally a morphological description of alterations in chromatin during interphase only roughly parallels the G1, S, and G2 phases defined by autoradiography following incorporation of tritiated thymidine. We suggest that both methods are valid through different ways of describing interphase.

Animals↗

PubMed1998

Application of interphase fluorescence in situ Hybridization for the detection of the Burkitt translocation t(8;14)(q24;q32) in B-cell lymphomas.

The translocation t(8;14)(q24;q32) is the characteristic chromosomal aberration of Burkitt's-type lymphomas and leukemias (BLs). On the molecular level, the t(8;14) juxtaposes the c-myc gene in 8q24 next to the IgH locus in 14q32, resulting in overexpression of the transcription factor c-Myc. The detection of a t(8;14) is a major aim in the diagnostic process of all patients with high-grade B-cell lymphomas because treatment strategies differ between BL and other high-grade lymphomas. As chromosome analyses are sometimes hampered by the low yield or poor quality of metaphase spreads and as the application of molecular genetic techniques is limited by the distribution of the 8q24 breakpoints over a region of about some hundred kilobases, we set out to establish an interphase fluorescence in situ hybridization (FISH) assay for the detection of the t(8;14). A cosmid probe hybridizing to the IgH constant region in 14q32 was combined with a differently labeled probe of pooled cosmid clones spanning the c-myc locus in 8q24. Interphase nuclei lacking a t(8;14) show two separated signals corresponding to each probe, whereas interphase nuclei carrying a t(8;14) display a split of the c-myc probe and a colocalization of at least one of the splitted signals with the IgH probe. Based on the results of extensive control studies, the cutoff level for this stringent (type I) criteria was set at 2%. Additionally, colocalization of at least one c-myc signal with one IgH signal alone (without signal split for the c-myc probe) was used as a less stringent (type II) criteria with a cutoff limit of 11%. Nine BLs and one Burkitt-like lymphoma were investigated by this approach. Cytogenetically, all tumors contained a translocation t(8;14)(q24;q32) except for one BL, in which cytogenetic analysis had failed. In interphase FISH, all lymphomas and leukemias met the less stringent criteria for the diagnosis of the t(8;14). Additionally, in all tumors but the Burkitt-like lymphoma, a t(8;14) could be diagnosed according to the stringent criteria. The percentage of cells found to harbor the t(8;14) by FISH ranged from 4.3% to 100%. Comparison of cytogenetic and FISH results revealed a significantly lower percentage of t(8;14)+ interphase nuclei than metaphase cells (P = .004). In conclusion, the described FISH assay provides a feasible and sensitive tool for the routine detection of the translocation t(8;14) in interphase cells which might also offer new insights into the biology of high-grade B-cell lymphomas.

Adult↗

PubMed1972

Induction of prophase in interphase nuclei by fusion with metaphase cells.

Fusion of an interphase cell with a metaphase cell results in profound changes in the interphase chromatin that have been called "chromosome pulverization" or "premature chromosome condensation" In addition to the usual light microscopy, the nature of the changes has been investigated in the present study with electron microscopy and biochemical techniques Metaphase and interphase cells were mixed and fused at 37 degrees C by means of ultraviolet-inactivated Sendai virus. After cell fusion, morphological changes in interphase nuclei occurred only in binucleate cells which contained one intact set of metaphase chromosomes Irrespective of the nuclear stage at the time of cell fusion, the morphologic changes that occurred 5-20 min later simulated very closely a sequence of events that characterizes the normal G(2)-prophase transition. Radioautography revealed that, late in the process, substantial amounts of RNA and probably protein were transferred from the interphase nucleus into the cytoplasm of fused cells. Thus, the findings indicate the existence in metaphase cells of factor(s) which are capable of initiating biochemical and morphological events in interphase nuclei intrinsic to the normal mitotic process.

Animals↗

PubMed1977

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation.

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.

Cell Division↗

PubMed1991

Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.

We have used the proximity of probe hybridization sites in interphase chromatin to derive the order of DNA sequences in a 2-3-Mbp region of human chromosome Xq28. The map generated bridges the results of genetic and pulsed-field gel electrophoresis mapping to produce a more complete map of Xq28 than possible with either of these other techniques alone. Two-color fluorescence in situ hybridization (FISH) was used to detect the positions of two or more probes in G1 male interphase nuclei. We show that cosmids that are 50 kbp to 2-3 Mbp apart can be ordered rapidly with two alternative approaches: (1) by comparing the average measured distance between two probes and (2) simply by scoring the order of red and green fluorescent dots after detection of three or more probes with two fluorochromes. The validity of these approaches is demonstrated using five cosmids from a region spanning approximately 800 kbp that includes the factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and color-vision pigment (CV) genes. The cosmid map derived from interphase mapping is consistent with the map determined by restriction-fragment analysis. The two interphase mapping approaches were then used (1) to orient the F8/CV cluster relative to two markers, c1A1 and st14c, which we show by metaphase mapping to be proximal to the F8/CV cluster, (2) to position st14c (DXS52) between c1A1 and F8, and (3) to orient the CV gene cluster relative to G6PD by using two CV-flanking cosmids, 18b41 and fr7. The probe order in Xq28 derived from interphase proximity is cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel. We also show that, to determine their order by using metaphase chromosomes, sequences must be at least 1 Mbp apart, an order of magnitude greater than required in interphase chromatin. The data show that FISH mapping is a simple way to order sequences separated by greater than or equal to 50 kbp for the construction of long-range maps of mammalian genomes.

Chromosome Mapping↗

PubMed1990

Detection of monosomy 7 in interphase cells of patients with myeloid disorders.

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.

Chromosome Deletion↗

PubMed1994

Microtubule converging centers and reorganization of the interphase cytoskeleton and the mitotic spindle in higher plant Haemanthus.

We analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plant Haemanthus. In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either "free" or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus. Elevated temperature (35 degrees-37 degrees C) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase-interphase transition and in some interphase cells, indicating temperature and stage dependence. Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle in Haemanthus endosperm.

Cell Cycle↗

PubMed1995

Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro.

Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

Animals↗

PubMed1995

Prenatal diagnosis of trisomy 21 using interphase fluorescence in situ hybridization of post-replicated cells with site-specific cosmid and cosmid contig probes.

Interphase fluorescence in situ hybridization (FISH) with chromosome 21-specific cosmid clones was used to identify trisomy 21 in cultured and uncultured amniotic cells. Two novel site-specific cosmid clones (regions 21q22 and 21qtel) were compared with a cosmid contig (Zheng et al., 1992). Correct identification of chromosome 21 copy number was made in 65-75 per cent of trisomic cells and in 70-75 per cent of normal disomic cells by using all the tested probes. However, the chromosome 21-specific telomeric probe (cos 17F8) showed the best results due to more intense and clearly visible hybridization. Utilization of a directly fluorophorated telomeric probe using Cy3-dCTP and FluorX-dCTP allows accurate detection of chromosome 21 in a fast 'one-step' FISH procedure on uncultured interphase nuclei. In addition, we compared the efficacy of FISH analysis for the total population of interphase cells and cells in the post-replication (late S, G2) periods of the cell cycle. Selective scoring of cells in the post-replicative period (showing a pair of hybridization signals on each chromatid of the replicated interphase chromosome) increased the number of informative nuclei by up to 95-97 per cent. This approach allows cells with overlapping chromosomes, artificial double hybridization signals on separate chromatids in interphase chromosomes, background hybridization, and polyploid cells to be analysed. Application of directly labelled telomeric cosmid probes and integral analysis of hybridized nuclei in the pre- and post-replication periods of the cell cycle may help to further improve the prenatal detection of trisomy 21.

Amniotic Fluid↗

PubMed2003

Residual risk for cytogenetic abnormalities after prenatal diagnosis by interphase fluorescence in situ hybridization (FISH).

Results from conventional cytogenetic studies on 21 609 amniotic fluid specimens were analyzed retrospectively to determine the residual risk for a cytogenetic abnormality if interphase FISH, capable of only detecting aneuploidy for chromosomes 13, 18, 21, X and Y, was performed and did not reveal an abnormality. Detection rates (the probability of detecting a cytogenetic abnormality when an abnormality is present) and residual risks (the likelihood of a cytogenetic abnormality, in view of normal interphase FISH results) were calculated for the four major clinical indications for prenatal diagnosis (advanced maternal age, abnormal maternal serum screen indicating increased risk for trisomy 18 or trisomy 21, abnormal maternal serum screen indicating increased risk for neural tube defects and ultrasound abnormality). Differences in detection rates were observed to depend on clinical indication and presence or absence of ultrasound abnormalities. The detection rate ranged from 18.2 to 82.6% depending on the clinical indication. The detection rates of abnormalities significant to the pregnancy being evaluated (i.e. abnormalities excluding familial balanced rearrangements and familial markers) were between 28.6 and 86.4%. The presence of ultrasound abnormalities increased the detection rate from 72.2 to 92.5% for advanced maternal age and from 78.6 to 91.3% for abnormal maternal serum screen, indicating increased risk for trisomy 18 or trisomy 21. With regard to residual risk, the risk for a clinically significant abnormality decreased from 0.9-10.1%, prior to the interphase FISH assay, to a residual risk of 0.6-1.5% following a normal interphase FISH result in the 4 groups studied. Providing patients with detection rates and residual risks, most relevant to their situation (clinical indication and presence or absence of ultrasound abnormality) during counseling, could help them better understand the advantages and limitations of interphase FISH in their prenatal diagnostic evaluation.

Adult↗

PubMed1994

Interphase AgNOR quantity is not related to DNA content in 11 established human cancer cell lines.

The relationship between interphase AgNOR quantity and DNA ploidy was evaluated in 11 cell lines derived from human tumors of different origin. Interphase AgNOR quantity was measured by morphometric image analysis and DNA content by cytofluorometry. The mean interphase AgNOR area ranged from 5.56 to 21.12 microns 2 and DNA ploidy from 2.05 to 3.30c. The interphase AgNOR value and DNA content were not significantly related (r = 0.23, P = 0.52). No significant correlation was found between DNA content and cell doubling time, whereas a strict relationship was confirmed to exist between interphase AgNOR quantity and rapidity of cell proliferation.

Cell Division↗

PubMed1994

Effect of telomeres on the interphase location of adjacent regions of the human X chromosome.

Chromosomes occupy specific nonrandom domains in the interphase nucleus of eukaryotic cells. We have used a Chinese hamster-human somatic cell hybrid line containing a single human X chromosome to study the interphase distribution of the Xp telomere using fluorescent in situ hybridization and optical sectioning. A derivative cell line in which the X chromosome has been broken at Xq22-24 and healed by the addition of cloned human telomeric sequences was also studied to determine if introduction of these sequences at a previously interstitial site changed its location in interphase. The endogenous Xp telomere occupies a specific, nonrandom, internal domain. Introduction of a telomere at a previously interstitial site did not alter the interphase nuclear location of that site. The results suggest that nonrandom interphase location of telomeres may not be determined solely by the DNA sequence of the telomere.

Cell Nucleus↗

PubMed1994

In vitro investigations of interphase and metaphase argyrophilic nucleolar organizer regions and cellular proliferation in the human urothelial cancer cell line HOK-1.

Detailed investigation of cell growth and nucleolar organizer region associated argyrophilic proteins (Ag-NORs) is necessary to assess a possible impact of Ag-NOR quantification on the diagnosis and prognosis of tumours. In this study, cellular proliferation of the transitional-cell carcinoma cell line HOK-1 was modulated over a period of 11 days by starvation and subsequent medium addition. Proliferation was determined daily by DNA flow cytometric estimation of S-phase fraction (SPF) and mitotic index (MI) calculation. The number and area of interphase Ag-NORs were quantified by automated image analysis daily and the number of Ag-NOR bearing chromosomes in metaphase was counted. In interphase nuclei, Ag-NOR area showed a highly significant correlation with SPF (p < 0.0001) whereas interphase Ag-NOR number showed significant correlation with MI (p < 0.05). A positive relationship between the number of Ag-NOR bearing chromosomes in metaphases and cellular proliferation was also observed. There is variability in Ag-NOR quantity during interphase and metaphase depending on growth conditions in vitro. Correlations of the number of interphase Ag-NORs with the MI on one hand and Ag-NOR area with SPF on the other provide further evidence that distribution and quantity of Ag-NORs are strongly influenced by the cell cycle phase within the structural-functional unit of the nucleolus.

Carcinoma, Transitional Cell↗

PubMed1985

Individual interphase chromosome domains revealed by in situ hybridization.

The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 micron wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.

Animals↗

PubMed1987

The blocked pinocytic activity of mitotic cells is restored in mitotic-interphase hybrids.

During mitosis there is an abrupt inhibition of a wide range of membrane functions, including fluid-phase and adsorptive pinocytosis. We have used cell hybrids formed between mitotic and interphase cells to approach the mechanism of this inhibition. We report that fluid pinocytosis is reactivated in the mitotic partner of hybrids formed between mitotic and interphase Chinese hamster ovary (CHO) cells. It thus appears that the interphase cell provides some necessary element(s) for membrane activity during mitosis. This dominance of interphase membrane properties stands in contrast with earlier evidence that mitotic nuclear properties dominate in similar mitotic-interphase hybrids.

Animals↗

PubMed1992

Longitudinal cytogenetic study of metaphase and interphase cells in childhood monosomy 7 syndrome.

A 15-year-old male with myelodysplastic syndrome (MDS) characterized by monosomy 7 was cytogenetically evaluated by metaphase karyotyping and fluorescence in situ hybridization (FISH) of interphase cells at six different points during the course of his disease. At diagnosis, there was complete agreement between metaphase and interphase findings. Interphase analysis alone provided important cytogenetic information on the first specimens received following intensive combination chemotherapy and bone marrow transplantation where metaphase analyses were uninformative. The detection of a minor post-treatment monosomy 7 population by interphase but not metaphase studies may have identified minimal residual disease prior to recurrence of MDS. From this longitudinal study, it is concluded that metaphase and interphase cytogenetic analyses form complementary approaches and that use of both provides greater analytical power when appropriate chromosome markers are available.

Adolescent↗

PubMed1993

Interphase FISH and morphologic analysis of AML.

Interphase cytogenetic analysis of peripheral white blood cells from a patient with acute myelogenous leukemia (AML) who was previously diagnosed with trisomy 8 was performed for the purpose of documenting clonal response to intensive chemotherapy. DNA in situ hybridization was performed using a probe specific for chromosome 8 alpha-satellite DNA sequences. Cells were stained with Wright stain prior to interphase analysis and photographed to allow correlation of cell morphology with abnormal karyotype. Prior to chemotherapy, the patient's leukocyte differential contained 8% blasts; interphase analysis revealed 23% trisomy 8 cells, including mature granulocytes. Forty days after the start of chemotherapy, at which point the patient had attained clinical remission, interphase analysis revealed only 2% cells with three signals, which was not statistically significant when compared with our own series of controls. The use of interphase FISH analysis in this case provides additional evidence that some leukemic blasts may be capable of limited differentiation in vivo and also suggests a differential sensitivity to chemotherapy between cytogenetically normal and abnormal hematopoietic precursor cells.

Aged↗