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PubMed1995

Interphase and metaphase detection of the breakpoint of 14q32 translocations in B-cell malignancies by double-color fluorescence in situ hybridization.

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24-68, containing VH segments. CY24-68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24-68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24-68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.

Burkitt Lymphoma↗
PubMed1995

Detection of chromosomal breakage in the 1cen-1q12 region of interphase human lymphocytes using multicolor fluorescence in situ hybridization with tandem DNA probes.

A novel multicolor fluorescence in situ hybridization approach, using an alpha satellite probe which labels the centromeric region on chromosome 1 and a classical satellite probe which targets an adjacent breakage-prone region (1q12), has been used to detect both hyperdiploidy and chromosome breakage in interphase human cells. With the use of this technique significant increases in chromosomal breakage were observed in interphase and metaphase lymphocytes irradiated in vitro. Metaphase analysis indicated that a significant proportion of these breakage events represented potentially stable aberrations such as translocations and inversions. A comparison of frequencies using a single classical satellite probe and the adjacent alpha and classical satellite probes indicated that this tandem label procedure allowed chromosomal breakage to be detected and distinguished from hyperdiploidy in untreated interphase lymphocytes, indicating the potential of this procedure for human biomonitoring. To determine whether this hybridization approach could detect alterations in humans, peripheral blood lymphocytes were obtained from a group of pesticide applicators and mixers and compared with a nonexposed control group. Significant increases in both hyperdiploidy and chromosomal breakage affecting the labeled region on chromosome 1 were observed in the pesticide-exposed group. These results indicate that this hybridization strategy allows hyperdiploidy and chromosomal breakage to be detected rapidly in interphase human cells and may facilitate the detection of chromosomal alterations in human populations exposed to carcinogenic and genotoxic agents using tissues which have not been previously amenable for cytogenetic analysis.

Cells, Cultured↗
PubMed1993

Mitosis-promoting factor activity of inducer mitotic cells may affect radiation yield of interphase chromosome breaks in the premature chromosome condensation assay.

We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of premature chromosome condensation to study the yield and the repair kinetics of radiation damage in interphase chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of premature chromosome condensation, uncovered two times more interphase chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to chromosome breaks, and subsequently to other types of lethal chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.

Amino Acid Sequence↗
PubMed1997

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

We have recently developed a method to detect tumor-specific rearrangement of the IgH gene in interphase nuclei by fluorescence in situ hybridization. Tumor-specific IgH gene rearrangement is equivalent to 14q32.33 translocation. Using this approach, we detected 14q32.33 translocation in 29 of 70 patients with B-cell non-Hodgkin's lymphoma (NHL). Chromosome t(3;14) was found in 10 of these 29 patients, and were demonstrated as a fusion signal of BCL6 and VH gene probes in interphase nuclei. Furthermore, in another series of 11 patients and a NHL cell line, we demonstrated t(14;18) and t(11;14) in interphase and metaphase cells with a combination of BCL2 (or PRAD1) with IgH gene probes. Interphase FISH with lymphoma-associated gene probes is a rapid procedure for cytogenetic diagnosis of B-cell NHL.

Cell Nucleus↗
PubMed1997

Spatial association of prekinetochores and chromocentres in the interphase nuclei of mouse cultured fibroblasts.

The structure and spatial distribution of prekinetochores and chromocentres (centromeric heterochromatin) in the interphase nuclei of mouse fibroblasts were studied by light and electron microscopy. Using the autoimmune serum GS reacting with centromeric proteins CENP-A, B, C in mammalian cells, we showed that most interphase prekinetochores were associated with chromocentres. During the interphase S period, including DNA replication in chromocentres, the macroorganization of chromocentres and their association with prekinetochores were maintained. Using cryosections and the electron microscopy immunogold technique, we found that centromeric proteins were mainly located at the chromocentre periphery. Nevertheless, we failed to find any definite kinetochore structures including those usually observed during mitosis at the sites of protein binding. Only small associates of fibrous material, presumably, corresponding to centromeric proteins, could be observed in association with the interphase mouse chromocentres in both cryosections and conventionally fixed sections.

Animals↗
PubMed1998

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

Adult↗
PubMed1975

Interphase chromosome arrangement in Anopheles atroparvus.

The arrangement of chromosomes in interphase nuclei of Anopheles atroparvus has been inferred from an analysis of: 1. The early stages of mitosis as seen following Quinacrine staining, and 2. The reversible effects on the chromatin pattern obtained following the treatment of living cells with various NaCl solutions, and the following conclusions have been reached: (a) The chromatin is connected to the nuclear membrane, (b) Homologous chromosomes show close side-by-side somatic pairing, (c) The long arms of the sex chromosomes form a fluorescent peripheral body, (d) The autosomes are strongly reflexed at the centromeres, (e) The autosomal centromeric regions are polarized towards the peripheral body, (f) The telomeric regions of all the autosomes are closely apposed.--A ring-shaped pattern of interphase chromatin is constantly and reversibly induced by NaCl 0.15 to 0.18 M solutions.--These relationships indicate a peripheral arrangement of the interphase somatic complement.--The distribution of the chromosomes in polytene nuclei and at the beginning of meiosis resembles that suggested above for somatic interphase cells. This distribution may apply more widely in the Diptera.

Animals↗
PubMed1994

Involvement of erythrocytic and granulomonocytic lineages by trisomy 11 in two cases of acute myelomonocytic leukemia with trilineage myelodysplasia. An interphase cytogenetic study.

To study the cytologic profile and lineage involvement in acute myeloid leukemia (AML) with trisomy 11, cytologic, cytogenetic, and interphase cytogenetic studies were performed at presentation in two cases of acute myelomonocytic leukemia (AML-M4). Patient 1 had +11 as the sole chromosome aberration in 16/20 karyotypes whereas two related clones with +11 in all abnormal metaphases (14/18) were detected in patient 2. A proportion of interphase cells with three signals, comparable to the proportion of abnormal metaphases, was detected by fluorescent in situ hybridization (FISH) in these patients. Morphologic aberrations of the nonblast cell population affecting multiple cell lineages, along with a circulating minor megakaryoblastic component, were observed at diagnosis in both patients. By separation of bone marrow cells over a density gradient of Percoll two cell fractions were obtained, the former containing more than 80% erythroid precursors (collected at a density of 1065-1075 mg/ml), the latter containing more than 78% blast cells plus granulomonocytic precursors (collected at a density of 1060-1055 mg/ml). FISH documented the presence of a majority of interphase nuclei with three signals in the erythroblast-enriched cell fraction and in the blast-enriched cell fraction. It is concluded that cytologic features, as well as interphase cytogenetic findings on enriched cell fractions, suggest the occurrence of multipotent stem cell involvement in AML-M4 with +11.

Adult↗
PubMed1994

Molecular distributions in interphases: statistical mechanical theory combined with molecular dynamics simulation of a model lipid bilayer.

A mean-field statistical mechanical theory has been developed to describe molecular distributions in interphases. The excluded volume interaction has been modeled in terms of a reversible work that is required to create a cavity of the solute size against a pressure tensor exerted by the surrounding interphase molecules. The free energy change associated with this compression process includes the configuration entropy as well as the change in conformational energy of the surrounding chain molecules. The lateral pressure profile in a model lipid bilayer (30.5 A2/chain molecule) has been calculated as a function of depth in the bilayer interior by molecular dynamics simulation. The lateral pressure has a plateau value of 309 +/- 48 bar in the highly ordered region and decreases abruptly in the center of the bilayer. Model calculations have shown that for solute molecules with ellipsoidal symmetry, the orientational order increases with the ratio of the long to short molecular axes at a given solute volume and increases with solute volume at a given axial ratio, in accordance with recent experimental data. Increased lateral pressure (p perpendicular) results in higher local order and exclusion of solute from the interphase, in parallel with the effect of surface density on the partitioning and local order. The logarithm of the interphase/water partition coefficient for spherical solutes decreases linearly with solute volume. This is also an excellent approximation for elongated solutes because of the relatively weak dependence of solute partitioning on molecular shape. The slope is equal to (2p perpendicular - p parallel)/3KBT, where p parallel is the normal pressure component, and different from that predicted by the mean-field lattice theory. Finally, the lattice theory has been extended herein to incorporate an additional constraint on chain packing in the interphase and to account for the effect of solute size on partitioning.

Biomechanical Phenomena↗
PubMedDate not supplied

Atomistic modeling of interphases in spider silk fibers.

We present a structural description of the intercrystalline polymer interphase based on atomistic modeling for spider dragline silk fibers. In the model small crystallites are connected by interphases. The interphases are assumed to be thin layers (< 4 nm) of statistically arranged chains. The calculated mechanical moduli of these interphases are significantly greater than the isotropic amorphous material.

Animals↗
PubMed2000

DNA in situ hybridization (interphase cytogenetics) versus comparative genomic hybridization (CGH) in human cancer: detection of numerical and structural chromosome aberrations.

DNA in situ hybridization techniques for cytogenetic analyses of human solid cancers are nowadays widely used for diagnostic and research purposes. The advantage of this methodology is that it can be applied to cells in the interphase state, thereby circumventing the need for high-quality metaphase preparations for karyotypic evaluation. In situ hybridization (ISH) with chromosome specific (peri)centromeric DNA probes, also termed "interphase cytogenetics", can be used to detect numerical changes, whereas comparative genomic hybridization (CGH) discloses chromosomal gains and losses, i.e. amplifications and deletions. We wanted to compare both methods in human solid tumors, and for this goal we evaluated ISH and CGH within a set of 20 selected prostatic adenocarcinomas. Chromosomes 7 and 8 were chosen for this analysis, since these chromosomes are frequently altered in prostate cancer. ISH with chromosome 7 and 8 specific centromeric DNA probes was applied to standard, formalin-fixed and paraffin-embedded, histological sections for numerical chromosome analysis. CGH with DNA's, extracted from the same histologic area of the archival specimens, was used for screening of gains and losses of 7 and 8. ISH with centromeric probes distinguished a total of 26 numerical aberrations of chromosome 7 and/or 8 in the set of 20 neoplasms. In the same set CGH revealed a total of 35 losses and gains. CGH alterations of 7 and 8 were seen in twenty-two of the 26 chromosomes (85%) that showed aberrations in the ISH analysis. Concordance between ISH and CGH was seen in 11 (of 26; 42%) chromosomes. Eight chromosomes were involved in gains (5 x #7, 3 x #8), three in losses (3 x #8). This included both complete (3/11) and partial (8/11) CGH confirmation of the numerical alteration. Partial CGH confirmation was defined as loss or gain of a chromosome arm with involvement of the centromeric region. In the majority of these cases it concerned a whole chromosome arm, mostly the long arm. We conclude that generally a fair correlation was found between ISH and CGH in interphase preparations of a series of prostate cancers. However, when specified in detail, most of the numerical ISH aberrations were only partly represented in the CGH analysis. On the one hand, it suggests that CGH does not adequately discriminate numerical abnormalities. On the other hand, it likely implies that not all numerical changes, as detected by interphase cytogenetics, are truly involving the whole chromosome. A part of these discrepancies might be caused by structural mechanisms, most notably isochromosome formation.

Adenocarcinoma↗
PubMed1971

Formation and distribution of nuclear pore complexes in interphase.

The possibility of nuclear pore formation in the interphase nucleus was investigated in control and phytohemagglutinin (PHA) stimulated lymphocytes by the freeze-etching technique. 48 hr after the addition of PHA, the newly formed blasts which had not as yet divided had at least twice the number of pores per nucleus as controls. This clearly demonstrates that in lymphocytes nuclear pore formation can take place during interphase. It has generally been assumed that the distribution of nuclear pore complexes in somatic animal cells is random. However, we have utilized freeze etched rat kidney cells and a computer program to evaluate pore distribution. We find a minimum pore center-to-center spacing of approximately 1300 A and multiples thereof with high frequency. This is strong evidence for a nonrandom distribution of nuclear pores. The nonrandomness may be related to an underlying chromosomal organization in interphase. Using three criteria for identifying prospective pore sites (membrane specialization, nonrandomness, and alteration of heterochromatin distribution), we have found forming pores in sectioned material from cultured human melanoma cells. While nuclear pore formation may take place in conjunction with reformation of the nuclear membrane, a mechanism also exists for their formation during interphase.

Animals↗
PubMed1974

Prophasing of interphase nuclei and induction of nuclear envelopes around metaphase chromosomes in HeLa and Chinese hamster homo- and heterokaryons.

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6-8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.

Cell Division↗
PubMed1973

Virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase Hep-2 cells infected with poliovirus.

Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed.

Carcinoma↗
PubMed2026

Live dynamics of induced cell-cell fusion between mitotic and interphasic cells.

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed the consequences of checkpoint override, forcing nuclei through untimely cell cycle transitions, and highlight how cell-cell fusion experiments can be a powerful system to study how competing cytoplasmic states are integrated in a shared cytoplasm, such as in muscle, placenta formation and in cancer.

Cell cycle↗
PubMed1986

Chromosome condensation activity in ovulated metaphase II mouse oocytes assayed by fusion with interphase blastomeres.

Fusion of large and small karyoplasts produced from metaphase II mouse oocytes with interphase blastomeres from 2-cell and 8-cell embryos (volume ratio of partners, 1:1) results in premature chromosome condensation (PCC) of the interphase nucleus in the majority of the fusion products (hybrids). Fused under the same experimental protocol, oocyte-derived cytoplasts also induce PCC of the blastomere nucleus in the fusion products (cybrids) provided they originate from recently ovulated oocytes (141/2-15 h after injection of human chorionic gonadotrophin (HCG)). In cytoplasts derived from older oocytes (16-20 h post-HCG) chromosome condensation activity gradually decreases with time as can be inferred from the increasing proportion of cybrids retaining interphase blastomere nuclei. However, even the oldest cytoplasts (19-20 h post-HCG) can induce PCC if the cytoplast volume significantly exceeds the volume of the interphase partner (7:1). We postulate that the condensation activity is predominantly bound to the nuclear apparatus (most probably to the chromosomes), and that in the cytoplasm of metaphase II mouse oocyte it decreases with post-ovulatory age.

Animals↗
PubMed1992

Interphase cytogenetics: a new tool for the study of genetic changes in brain tumors.

Interphase cytogenetics is the application of nonradioactive in situ hybridization with chromosome-specific DNA probes to interphase nuclei. In this study, interphase cytogenetics was used to investigate 66 primary brain tumors (33 gliomas, 30 meningiomas, and three medulloblastomas) for numerical chromosomal aberrations of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. Of the 33 gliomas (17 astrocytomas grades II, III, and IV, five oligoastrocytomas, seven oligodendrogliomas, and four ependymal tumors), 22 were near diploid, while the remaining 11 showed a significant triploid or tetraploid component. The predominant specific aberrations in gliomas were an over-representation of chromosome 7 (13 cases) and an under-representation of chromosome 10 (16 cases). These changes were observed in grade III and grade IV astrocytomas, as well as in oligodendrogliomas. Other frequent numerical changes were a gain of chromosome 17 (six cases) and a loss of chromosome 18 (seven cases). This loss of chromosome 18 seemed relatively specific for gliomas with an oligodendroglial component (six cases). Only two of 33 gliomas displayed no genetic abnormality with the probes used. Seven patients with astrocytomas died of their brain tumor during the clinical follow-up period. Their astrocytomas did not show a different chromosomal constitution compared to the other gliomas. For the meningiomas, the probe panel was extended with a probe specific for chromosome 22. Loss of chromosome 22 was obvious in 21 of the 30 meningiomas, and was the sole abnormality in 11 meningiomas; in the other 10, this loss was associated with other chromosomal changes. Five of these tumors with additional aberrations were recurrent or atypical meningiomas. It is suggested that interphase cytogenetics can contribute to a better understanding of the biological behavior of these tumors and possibly result in better insights into prognosis and strategies for therapy.

Adult↗
PubMed2001

[Detection of -5/5q- chromosome abnormality in myelodysplastic syndromes by interphase fluorescence in situ hybridization].

OBJECTIVE: To evaluate the value of interphase fluorescence in situ hybridization(FISH) in the detection of acquired complete and partial deletion of chromosome 5(-5/5q-) in myelodysplastic syndromes (MDS). METHODS: Marrow cells from 48 MDS patients and 10 normal controls were studied by interphase FISH using SpectrumRed directly labelled DNA specific probe for 5q31, 200 to 300 cells were scored for each case and cells with a red hybridization signal > 6% were defined as -5/5q- positive. RESULTS: In 48 MDS patients, 13 were positive for interphase FISH, of whom, 7 were positive and 6 were negative for conventional cytogenetics (CC). The difference in the percentage of positive cells detected by the two methods was statistically significant (P < 0.05). CONCLUSION: Interphase FISH is more sensitive than CC for the detection of -5/5q- in MDS.

Adult↗