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PubMed1976

Nucleolus degradation and growth induced by uv-microbeam irradiation of interphase cells grown in culture.

In contrast to total cell irradiation, local UV-microbeam irradiation can stimulate a significant diminution in the irradiated mature nucleoli in interphase mammalian cells in culture. This diminution is accompanied by the concomitant expansion of the unirradiated nucleoli within the same nucleus, and the total nucleolar volume per nucleus does not change appreciably. It is suggested that these nucleolar volume changes are the result of the dispersion, migration, and redistribution of the nucleolar material between competitive nucleolar organizer regions of the interphase nucleus.

Cell Division↗
PubMed2002

Interphase cytogenetic analysis of lymphoma-associated chromosomal breakpoints in primary diffuse large B-cell lymphomas of the central nervous system.

Primary central nervous system lymphomas (PCNSLs) are germinal center-derived diffuse large B-cell lymphomas (DLBCLs) arising in and remaining confined to the brain, the pathogenesis of which is poorly understood. We investigated 13 PCNSLs from immunocompetent patients by means of interphase cytogenetics on cryopreserved cells derived from stereotactic biopsies. Interphase fluorescence in situ hybridization (FISH) was performed for the detection of structural alterations affecting the IGH (14q32), IGK (2p12), IGL (22q11), BCL6 (3q27), MYC (8q24), CCND1 (11q13), MLT, and BCL2 (both 18q21) loci. Signal constellations indicating breakpoints within the IGH and IGK locus were detected in 5 and 1 PCNSLs, respectively. There was no evidence for a t(8;14), t(11;14), or t(14;18) in this series of tumors. Breakpoints in the BCL6 locus were observed in 3 of the 13 cases, and nuclear Bcl-6 protein expression was detected in 6 of 9 PCNSLs, including those with genomic alterations of the encoding locus. Gains of 18q21 represented the most frequent imbalances present in more than one third of all cases. Interestingly, these gains included the MLT gene. Thus, this study provides the first evidence for recurrent chromosomal translocations in PCNSLs. While they share similarities with extracerebral DLBCL with respect to the presence of IGH translocations, they appear to differ in the usage of translocation partner genes, which remain to be identified.

Adult↗
PubMed2004

Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.

Artifacts↗
PubMed1977

The structure of the human cell interphase chromatin in the mosaic form of Down's disease.

In an earlier publication we reported that, by using fluorescent microscopy with acridine orange (AO), we detected certain changes in the structure and function of interphase nuclear chromatin of peripheral blood lymphocytes of subjects with Down's disease (Fedorova, Inshakova and Spitkovsky, 1975). By employing the same method with additional thermal action on the cells studied it was shown that some of these changes were conditioned by the effect of the blood serum from a person with Down's syndrome (Fedorova and Spitkovsky, 1976). The purpose of the present work was to investigate the structure of the interphase chromatin of both disomic and trisomic human cells residing in the same serum, that is, in mosaic subjects. Small lymphocytes in the peripheral blood of mosaic mongols were selected for study, complete trisomic mongols and healthy subjects being used as controls.

Adolescent↗
PubMed1997

Chromosomal translocations in human soft tissue sarcomas by interphase fluorescence in situ hybridization.

In soft tissue sarcomas, clonal rearrangement of chromosomes has been shown by cytogenetic analysis to be unique and specific for tumor types. The development of fluorescence in situ hybridization (FISH) has allowed detection of chromosomal rearrangements in the interphase nuclei isolated from paraffin-embedded tissues. Three kinds of translocations in the interphase nuclei that were isolated from 47 cases of soft tissue sarcomas were examined by FISH with chromosome-specific DNA probes of centromeric and total probes. Of 47 soft tissue sarcomas 42 (89.4%) revealed tumor-specific translocations by retrospective cytogenetic analysis. Translocation t(X;18) was detected in 25/28 synovial sarcomas; translocation t(11;22) in 5/6 Ewing's sarcomas and primitive neuroectodermal tumors (PNET); and translocation t(12;16) was found in 12/13 liposarcomas, including 10 myxoid and two round cell types as clonal chromosomal aberrations specific for both subtypes. Based on the cytogenetic analysis, Ewing's sarcoma is related closely with PNET as shown by MIC2-protein reactivity. Other cytogenetic findings of translocation t(12;16) indicate that round cell liposarcomas share chromosomal changes with myxoid liposarcomas, and further suggest that both tumor subtypes of liposarcoma may possess common precursor cells. FISH is a useful aid in determining the tumor type of soft tissue sarcomas with regard to histogenetic origin.

Adolescent↗
PubMed1996

Assessment of intra-tumoral karyotypic heterogeneity by interphase cytogenetics in paraffin wax sections.

Aim-To analyse the effect of sectioning on the assessment of karyotypic heterogeneity by interphase cytogenetics in paraffin wax embedded normal squamous epithelium and to apply the principles derived to invasive cervical carcinoma.Methods-Normal male (n = 5) and female (n = 5) squamous epithelia were hybridised with peri-centromeric repeat probes specific for chromosomes X (DXZ1) and 17 (D17Z1) individually and in combination to assess the effect of sectioning on mono-, di-, tri-, and tetrasomic populations. Section thickness, interobserver variation and variation between different areas of the epithelium were evaluated. Invasive squamous carcinomas of the cervix (n = 5) were then hybridised with the DXZ1 probe and intratumoral heterogeneity was assessed by comparison of signal distributions obtained from different areas.Results-The optimum section thickness for the assessment of normal epithelium was 6 mum. Variation in the expected signal number in the range 1-4 did not introduce artefactual heterogeneity at this section thickness. The sensitivity of this approach for the detection of minor subpopulations was calculated to be 13-16%, 17-18% and 10-11% for mono-, tri- and tetrasomic populations, respectively. Karyotypic heterogeneity was detected in two of the five tumours and, in one case where the populations where clustered morphologically, a minor population representing 18% was identified.Conclusions-Interphase cytogenetic analysis of sections from paraffin wax embedded material can be used for the detection of minor subpopulations in tumours. This approach will be of particular value in the assessment of the relation between human papillomavirus infection and tumour karyotype and in the analysis of intraepithelial neoplasia.

Journal Article↗
PubMed2000

Application of CARD-ISH for Assessment of Numerical Chromosome Aberrations in Interphase Nuclei of Human Tumor Cells.

In this study based on the study of a centromeric DNA probe specific for chromosome 8 the authors standardized a method for the enzymatic detection of specific chromosome copy numbers on interphase nuclei from tumor tissue samples. Since the in situ hybridization (ISH) of chromosome 8 specific probe was revealed with a catalyzed reporter deposition (CARD), which allows a high amplification of the hybridization signal, the method was designated as CARD-ISH. This method has been standardized on interphase nuclei isolated from clinical samples of pituitary adenomas, as well as on human normal lymphocytes. On the same samples, they also evaluated chromosome 8 copy number distribution by FISH. Comparison between CARD-ISH and FISH results showed no significant differences between the two methods, proposing CARD-ISH as a reliable alternative to FISH for chromosome numerical aberration assessment in laboratories that do not have specific facilities for epifluorescence microscopy or cytogenetics and that need a long-term storage of slides that had been used for diagnostic purposes. Int J Surg Pathol 8(3):201-206, 2000

Journal Article↗
PubMed1993

Discrimination of distinct subpopulations within a tumor with combined double immunophenotyping and interphase cytogenetics.

We describe a method that enables detection and immunophenotypical characterization of distinct subpopulations within a cytogenetically defined tumor clone. Coexisting normal cells do not hinder microscopic evaluation because they can be distinguished from cytogenetically aberrant tumor cells. This is also true when normal and neoplastic cells cannot be clearly distinguished by cytology or immunohistochemistry, i.e., if both constituents have similar immunophenotypes and morphology. The method is based on fluorescence double staining for two different antigens combined with interphase cytogenetic analysis. It is referred to as "Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)." In a case of follicular lymphoma we demonstrate that FICTION can differentiate bcl-2-positive malignant and non-malignant cells and can verify the presence of bcl-2-positive but cytogenetically inconspicuous T-lymphocytes.

Antigens, CD↗
PubMedDate not supplied

A repetitive probe for FISH analysis of bovine interphase nuclei.

The purpose of this study was to generate repetitive DNA sequence probes for the analysis of interphase nuclei by fluorescent in situ hybridisation (FISH). Such probes are useful for the diagnosis of chromosomal abnormalities in bovine preimplanted embryos. Of the seven probes (E1A, E4A, Ba, H1A, W18, W22, W5) that were generated and partially sequenced, five corresponded to previously described Bos taurus repetitive DNA (E1A, E4A, Ba, W18, W5), one probe (W22) shared no homology with other DNA sequences and one (H1A) displayed a significant homology with Rattus norvegicus mRNA for secretin receptor transmembrane domain 3. Fluorescent in situ hybridisation was performed on metaphase bovine fibroblast cells and showed that five of the seven probes hybridised most centromeres (E1A, E4A, Ba, W18, W22), one labelled the arms of all chromosomes (W5) and the H1A probe was specific to three chromosomes (ch14, ch20, and ch25). Moreover, FISH with H1A resulted in interpretable signals on interphase nuclei in 88% of the cases, while the other probes yielded only dispersed overlapping signals.

Journal Article↗
PubMed1976

An ultrastructural and radioautographic study of the chromocentric interphase nucleus in plant meristematic cells (Raphanus sativus).

In Raphanus sativus, the mitotic chromosomes are quite short and, on reaching the cell poles, soon undergo extensive unravelling. By late telophase and early interphase, only a few chromosome segments, believed to correspond to the centromeric regions, are still visible in the form of chromocentres closely associated with the nuclear envelope. Although interphase nuclei show little internal structural differentiation, high-resolution radioautography has permitted us to establish which of them have reached the early, mid and late S periods. In early S nuclei, only the nucleolus and the euchromatin which pervades the nuclear cavity become labelled. By the mid S-period, the diffuse chromatin and nucleolus incorporate less thymidine and DNA synthesis is initiated within the peripheral chromocentres. Subsequently, the radioautographic grains become restricted to the chromocentres. The finding that certain late S nuclei exhibit loosely organized chromocentres strongly suggests that these heterochromatic chromosome segments undergo important conformational modifications during DNA replication. Finally, the presence of radioautographic grains over the lacunar regions of the nucleolus in early and mid S nuclei demonstrates that intranucleolar DNA replicates during the earlier portion of the S-period.

Autoradiography↗
PubMed2001

Comparison of F ratios generated from interphase and metaphase chromosome damage induced by high doses of low- and high-LET radiation.

Although biophysical models predict a difference in the ratio of interchromosomal to intrachromosomal interarm exchanges (F ratio) for low- and high-LET radiations, few experimental data support this prediction. However, the F ratios in experiments to date have been generated using data on chromosome aberrations in samples collected at the first postirradiation mitosis, which may not be indicative of the aberrations formed in interphase after exposure to high-LET radiations. In the present study, we exposed human lymphocytes in vitro to 2 and 5 Gy of gamma rays and 3 Gy of 1 GeV/nucleon iron ions (LET = 140 keV/micrometer), stimulated the cells to grow with phytohemagglutinin (PHA), and collected the condensed chromosomes after 48 h of incubation using both chemically induced premature chromosome condensation (PCC) and the conventional metaphase techniques. The PCC technique used here condenses chromosomes mostly in the G(2) phase of the cell cycle. The F ratio was calculated using data on asymmetrical chromosome aberrations in both the PCC and metaphase samples. It was found that the F ratios were similar for the samples irradiated with low- and high-LET radiation and collected at metaphase. However, for irradiated samples assayed by PCC, the F ratio was found to be 8.2 +/- 2.0 for 5 Gy gamma rays and 5.2 +/- 0.9 for 3 Gy iron ions. The distribution of the aberrations indicated that, in the PCC samples irradiated with iron ions, most of the centric rings occurred in spreads containing five or more asymmetrical aberrations. These heavily damaged cells, which were either less likely to reach mitosis or may reach mitosis at a later time, were responsible for the difference in the F ratios generated from interphase and metaphase analysis after exposure to iron ions.

Chromosome Aberrations↗
PubMed1998

CGH-detected DNA sequence copy number amplifications can be confirmed by interphase-FISH: new possiblities for prognostic approaches in oral squamous cell carcinomas.

In order to control the data obtained by comparative genomic hybridization (CGH) on DNA sequence copy number amplifications, 20 oral squamous cell carcinomas (SCC) were subjected to interphase fluorescence in situ hybridization (I-FISH) examination using specific DNA probes for the oncogenes int2 and erbB-2, and the corresponding centromeric probes of chromosomes 11 and 17. In all cases characterized by distinct peaks of the CGH profile on the critical chromosomal segment 11q13, these data could be clearly substantiated by the I-FISH analyses using the int2 probe and estimating the signal index, the int2/centromer 11 relation, and the fraction of nuclei with high int2 signal numbers. In addition, I-FISH detected smaller cell fractions with high signal numbers (and/or signal clusters) in some tumors which were not definitely conspicuous in CGH. In contrast to int2, erbB-2 amplification apparently does not play a major role in oral SCCs, as the blurred peaks of CGH profiles on chromosome 17ql 1.2-q12 corresponded well with the findings of I-FISH using the erbB-2 probe. Gain of a whole chromosome 17 is apparently a rather common feature of these tumors. In conclusion, the combination of interphase FISH with oncogene-specific probes and CGH is regarded as a valuable means of practical molecular cytogenetic analysis of oral SCCs which could eventually achieve high practical importance in the pathologic analysis of these tumors and in prognosis of their development.

Adult↗
PubMed1976

[Local unscheduled DNA synthesis following irradiation of a portion of mitotic chromosomes or interphase nucleus with UV microbeam].

Part of mitotic chromosome sets of the metaphase or anaphase SPEW mammalian cells were irradiated with an ultraviolet microbeam, and the subsequent incorporation of H3-thymidine into the postmitotic daughter nuclei was studied by autoradiography. The located areas of the unscheduled DNA synthesis were detected in these nuclei. This synthesis was likely to be due to repair process at the sites of postmitotic localization of the irradiated chromosomes. It is suggested that the selective chromosomal labeling induced by microirradiation could be employed in studying patterns of chromosome localization in the interphase nuclei. Local inhibition of the replicative DNA synthesis and local stimulation of the unscheduled DNA synthesis were observed after a partial microirradiation of the interphase nuclei. This unscheduled DNA synthesis was unaffected by hydroxyurea at concentration which inhibited normal DNA synthesis.

Cell Line↗
PubMed1976

[Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei].

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.

Animals↗
PubMed1999

Chemistry in Interphases-A New Approach to Organometallic Syntheses and Catalysis.

Combining the advantages of homogeneous and heterogeneous catalysis is still a problem that has not been satisfactorily solved. Chemistry in interphases offers a new approach for overcoming the difficulties, as is described in this article. Owing to the swellable or porous matrix, an interphase represents a state which in the most favorable case is similar to that of a solution. Moreover the proper choice of a mobile hybrid copolymer enables the control of the density and accessibility of the reactive centers, which results in a distinct improvement of the activity of the catalysts (two examples are shown schematically).

Journal Article↗
PubMed1999

[Pulmonary surfactant status in experimental dust-induced bronchitis based on the data from dynamic interphase tensiometry].

The disease was simulated by inhalation of dust containing 29% of free silica in Wistar male rats during 8 months. One group of the subjects was kept under 26-28 degrees C and other one--under 38-40 degrees C. Dynamic surface tension of lung tissue homogenate was studied by means of computer tensiometer MRT (Lauda, Germany). Interphase tensiometry precisely characterizes state of lung surfactant in experimental chronic dust bronchitis. Equilibrium surface tension and surface tension for 0.01 and 1 sec deteriorate during combined exposure to silica dust and heating microclimate. The studies demonstrated strong correlation between interphase tensiogram parameters and individual lipids content of lung surfactant. Surface tension is related to the state of lipids peroxidation in lungs and antioxidant systems.

Animals↗
PubMed2000

Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.

Interphase fluorescence in situ hybridization (FISH) studies of chromosomal region 13q14 were performed to investigate the incidence and clinical importance of deletions in multiple myeloma (MM). Monoallelic deletions of the retinoblastoma-1 (rb-1) gene and the D13S319 locus were observed in 48 of 104 patients (46.2%) and in 28 of 72 (38.9%) patients, respectively, with newly diagnosed MM. FISH studies found that 13q14 was deleted in all 17 patients with karyotypic evidence of monosomy 13 or deletion of 13q but also in 9 of 19 patients with apparently normal karyotypes. Patients with a 13q14 deletion were more likely to have stage III disease (P =.022), higher serum levels of beta(2)-microglobulin (P =.059), and a higher percentage of bone marrow plasma cells (P =.085) than patients with a normal 13q14 status on FISH analysis. In patients with a deletion of 13q14, myeloma cell proliferation (Ki-67) was markedly increased (22.0% +/- 6.9% compared with 15.6% +/- 8.2% in patients without the deletion; P =.0008). Evaluation of bromodeoxyuridine incorporation in 5 patients revealed that both rb-1-deleted and rb-1-normal MM subpopulations were proliferative. The presence of a 13q14 deletion on FISH analysis was associated with a significantly lower rate of response to conventional-dose chemotherapy (40.8% compared with 78. 6%; P =.009) and a shorter overall survival (24.2 months compared with > 60 months; P <.005) than in patients without the deletion. Multivariate analysis of prognostic factors confirmed the independent predictive value of 13q14 deletions for shortened survival. In conclusion, deletions of 13q14 are frequently detected by interphase FISH in patients with newly diagnosed MM, correlate with increased proliferative activity, and represent an independent adverse prognostic feature in MM. (Blood. 2000;95:1925-1930)

Adult↗
PubMed1999

Vinflunine, a new vinca alkaloid: cytotoxicity, cellular accumulation and action on the interphasic and mitotic microtubule cytoskeleton of PtK2 cells.

Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.

Animals↗