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PubMed2007

Discrete states of a protein interaction network govern interphase and mitotic microtubule dynamics.

The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.

Animals↗

PubMed2000

Computerized video time-lapse microscopy studies of ionizing radiation-induced rapid-interphase and mitosis-related apoptosis in lymphoid cells.

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.

Animals↗

PubMedDate not supplied

[Globular model of interphase chromosome and intrachromosomal exchange aberrations].

A globular folding model formerly proposed for large-scale interphase chromatin/chromosome organization is investigated here by computer simulation. Large-scale structure of interphase chromosome is suggested to be determined by volumetric interactions between chromosome subunits resulting in folding of subunit chain in a globular state. Structure unit of interphase chromosome is considered as a spherical structure consisting of a cluster of compact chromatin domains. These structure units/domain clusters were termed as a superdomains to emphasize their supradomain organization. Globular state of superdomain chain with excluded volume as well as phase transitions between condensed (globular) and decondensed (coiled) states are studied by dynamic Monte-Carlo approach. Globular model is supported by confocal microscopy data. It extends current view on interphase chromosome as a random Gauss polymer chain. Radiation-induced intrachromosome exchange aberrations (intrachanges) are modeled on the basis of the contact hypothesis. It implies that intrachanges result from radiation damage of chromatin mainly in contacted subunits. Computer modeling of chromosome structure provides a wealth of information about proximity effects and results in prediction of pattern of intrachromosome contacts. Calculated intrachange frequencies agree well with experimental data. Globular model suggests a physical mechanism for conformational changes of large-scale chromosome structure similar to phase transitions in polymer systems. These rearrangements of structure organization of chromosomes in the nuclei following irradiation could be mechanistically linked to the complex chromosome aberrations formation as well as to alteration of gene expression due to position variegation effect.

Chromatin↗

PubMed2001

Interphase FISH with chromosome-specific protelomere probes for rapid prenatal diagnosis in a reciprocal translocation carrier.

Interphase fluorescence in situ hybridization (FISH) analysis has become an accepted practice for rapid preliminary analysis of chromosome aneuploidy from direct amniocyte preparations. The use of dual-color interphase FISH analysis with chromosome-specific protelomere probes for the rapid exclusion of chromosomally unbalanced segregants in the pregnancy of a reciprocal translocation carrier is reported. Amniocentesis was performed at 16 weeks gestation on the carrier of a t(5;14)(p14.2;p13), who was ascertained after the birth of a son with the der(5) chromosome. Interphase FISH analysis with probes for 5pter, 5qter and 14qter showed two signals for each, consistent with alternate segregation of the maternal translocation. Subsequent metaphase analysis confirmed a 46,XY,t(5;14)(p14.2;p13)mat karyotype in the fetus. This case illustrates the utility of interphase FISH analysis with protelomere probes for rapid prenatal diagnosis in cases of parental reciprocal translocation.

Adult↗

PubMed2000

[Detection of trisomy 8 with interphase fluorescence in situ hybridization in myelodysplastic syndromes].

OBJECTIVE: To explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of trisomy 8 in myelodysplastic syndromes (MDS). METHODS: Conventional cytogenetics (CC) and interphase FISH using SpectrumGreen labelled chromosome 8 centromere specific probe were simultaneously carried out to detect trisomy 8 in 69 MDS and 6 normal individuals. RESULTS: Two hundred interphase cells were counted and cells with three green hybridization spots > 3% was assigned. Eleven cases displayed trisomy 8 by CC and were confirmed in 10 by FISH. In 7 cases, the percentage of trisomy 8 cells was significantly lower by FISH than by CC. Seven cases displayed trisomy 8 by FISH in 58 cases who did not show trisomy 8 by CC. Of the 7 cases, two had 2 and 3 marker chromosomes respectively, 4 had normal karyotypes. CONCLUSIONS: Interphase FISH was a useful method for the detection of trisomy 8 in MDS, especially in patients with normal karyotype or marker chromosome. It was a important complement to CC.

Adult↗

PubMed2002

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis.

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.

Amino Acid Sequence↗

PubMedDate not supplied

Interphase cytogenetics of hematological neoplasms under the perspective of the novel WHO classification.

The novel WHO classification of tumors of hematopoietic and lymphoid tissues highlights the importance of genetic aberrations for the proper diagnosis of these malignancies. Among those techniques allowing detection of chromosomal changes, we have shown repeatedly that interphase fluorescence in situ hybridization (FISH) is currently the most robust and reliable technique for the diagnosis of lymphoma-associated translocations. Interphase FISH assays for the detection of the most frequent chromosomal alterations in myeloid and lymphoid disorders have become commercially available during the recent past. The sensitivity of some of these assays reaches levels below 1% providing valuable tools not only for diagnosis but also for follow-up and detection of minimal residual disease. Improvements of interphase FISH include the development of multicolor interphase FISH assays to detect several of the diagnostic changes in a single cell and of simultaneous fluorescence immunophenotyping (FICTION technique) to correlate phenotypic and genotypic cell features.

Hematologic Neoplasms↗

PubMed1991

Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes.

Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.

Acute Disease↗

PubMed1990

Radiation-induced interphase death observed in human T-cell lymphoma cells established as a nude mouse tumor line.

Interphase death of cells occurs physiologically in healthy animal tissues as well as in tissues pathologically injured by radiation or drugs. An active self-destruction process has been found to play a major role in the interphase death of highly radiosensitive cells. However, the mechanism of this radiation-induced interphase death in human lymphoma has not yet been studied in detail. In the present study, we examined a lymphoma derived from a child lymphoblastic lymphoma bearing CD1, CD4, and CD8 antigens and established in nude mice. Low-dose x-irradiation of this lymphoma induced interphase cell death with characteristic morphological and biological changes of an active self-destruction process, i.e., changes in cell surface appearance seen using scanning electron microscopy and nuclear fragmentation accompanied with an increase in free DNA. The process was proved to require protein synthesis. It was concluded that the radiosensitivity of this T-cell lymphoma of common thymic type is mainly due to the occurrence of the active self-destruction process.

Animals↗

PubMed1989

Relationship between interphasic nucleolar organizer regions and growth rate in two neuroblastoma cell lines.

The relationship between the quantity of silver-stained interphasic nucleolar organizer regions (NORs) and nuclear synthetic activity, caryotype, and growth rate was studied in two established neuroblastoma cell lines (CHP 212 and HTB 10). Statistical analysis of silver-stained NORs revealed four times as many in CHP 212 cells compared with HTB 10 cells. No difference was observed in the ribosomal RNA synthesis between the two cell lines. The caryotype index was 1.2 for CHP 212 and 1.0 for HTB 10 cells. The number of chromosomes carrying NORs and the quantity of ribosomal genes was found to be the same for the two cell lines. Doubling time of CHP 212 cells was 20 hours compared with 54 hours for HTB 10 cells. In CHP 212 cells bindering of cell duplication by serum deprivation induced a progressive lowering (calculated at 48, 72, and 96 hours) of the quantity of silver-stained interphasic NORs. Recovery of duplication by new serum addition induced, after 24 hours, an increase of the quantity of silver-stained interphasic NORs up to control levels. In the light of available data, these results indicate that the quantity of interphasic NORs is strictly correlated only to the growth rate of the cell.

Cell Line↗

PubMed1987

[Biological effect of HPD photosensitivity on human stomach cancer cells (MGC-803) in mitotic phase and interphase].

Photodynamic effect of HPD plus light on human stomach cancer cells (MGC-803) in the mitotic phase and interphase was studied. The synchronized mitotic cells were selected from the monolayer of asynchronous growing cells by shaking the culture flask. It was found that the killing frequency of HPD photosensitization was markedly different in the mitotic phase and interphase of synchronous growing MGC-803 cells, but a very small difference was found in HPD alone or light alone groups. The results show that, for adhesive cells in the two phases, there was a marked difference in damage pattern and recovery rate of mitosis 36 hr, 60 hr and 108 hr after photoradiation. The tolerance of mitotic cells to HPD plus light was much greater than that of the interphase cells. In the interphase, a great many of nude nuclei were seen and in the mitotic phase, cytokinesis was arrested resulting in many binucleated or multinucleated cells.

Cell Cycle↗

PubMed1980

[Isolation and comparative characteristics of the DNA attached to the axial structures of interphase nuclei and metaphase chromosomes].

DNA fractions enriched in sequences located closely to the sites of attachment of DNA loops to the axial protein structures of interphase nuclei ad metaphase chromosomes were isolated with the aid of two techniques. The method involving treatment with EcorI endonuclease has been described earlier. The new method is based on the mild treatment of the preparations with micrococcal nuclease. The latter method allows one to obtain the fraction of attached DNA of different length and to compare their properties. The average distance between two attachment sites was measured. It is equal to about 6.10(4) base pairs in the both interphase nuclei and metaphase chromosomes. The attached DNA obtained with the aid of micrococcal nuclease treatment is not enriched in satellite but contains a high amount of middle repetitive sequences renaturing in the Cot interval from 10(-2) to 10(0). According to renaturation properties and distribution in CsCl density gradient the fractions of attached DNA of similar size from metaphase chromosomes and from interphase nuclei are virtually identical. The data obtained indicate that the DNA sequences involved in the attachment of DNA loops to the matrix of interphase nucleus are the same as in the case of metaphase chromosomes.

Animals↗

PubMed1995

Induction and rejoining of DNA double-strand breaks and interphase chromosome breaks after exposure to X rays in one normal and two hypersensitive human fibroblast cell lines.

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Cell Line↗

PubMed1995

Higher reproducibility of morphometric analysis over the counting method for interphase AgNOR quantification.

In a series of 40 breast carcinomas, the reproducibility of two different methods for interphase AgNOR quantification was evaluated. Two operators independently defined on each slide the interphase AgNOR quantity both by measuring the area of the silver-stained structures using image cytometry and counting the AgNOR number directly at the microscope. The correlation between the values obtained by the two observers was statistically significant, but the correlation coefficient between AgNOR areas (r = 0.79; P < 0.001) was greater than that between AgNOR numbers (r = 0.38; P = 0.014). On the other hand, when interphase AgNOR area and number values obtained by each observer were compared, no significant correlation was found. This study has demonstrated that the two different methods for interphase AgNOR quantification are not comparable, and that morphometric analysis is more objective and reproducible than the counting method.

Humans↗

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↗