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PubMed1997

Detection of karyotype changes in interphase cells: oligonucleotide-primed in situ labelling versus fluorescence in situ hybridization.

Interphase cytogenetics is a rapidly developing technique which is usually performed by fluorescence in situ hybridization (FISH). Recently, oligonucleotide-primed in situ synthesis (PRINS) has become established as a method of labelling centromeric regions of chromosomes in metaphase spreads. We tested the suitability of PRINS in detecting the exact copy number of chromosomes 1, 3, 7 and 8 in intact interphase cells of 17 cytological preparations derived from normal and neoplastic tissues. Control procedures consisted in preparation of metaphase spreads of lymphocytes of healthy donors, conventional cytogenetics in some of the specimens, and omission of the primers or Taq polymerase from the reaction mixture. All specimens were additionally examined by FISH and analysed blind by two experienced observers. Both PRINS and FISH revealed a corresponding distribution of hybridization signals for all chromosomes examined in specimens of normal bone marrow (n = 5), normal liver cells (n = 5), three samples of acute nonlymphocytic leukaemia in which conventional chromosome analyses had shown monosomy 7 or trisomy 8, and in four hepatocellular carcinomas that displayed trisomy 1. Overall, statistical analysis revealed no significant difference in the signal distribution between the two techniques. Our results demonstrate that PRINS is as reliable as FISH for detecting chromosome copy numbers in interphase nuclei of intact cells. The PRINS method, however, is easier to perform, faster and less expensive, holding great potential for future applications in diagnostic pathology.

Biopsy↗
PubMed2000

Multilocus genetic analysis of single interphase cells by spectral imaging.

Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types.

Blastomeres↗
PubMed1985

Degree of conservation of HeLa interphase nonhistone antigens in metaphase and with chromatin from non-human cells.

Immunological procedures were applied to determine the degree of conservation of the nonhistone proteins of HeLa interphase chromatin. Polyacrylamide gels were overlaid with antiserum to HeLa interphase chromatin, and 125I-Protein A was used to detect bound antibodies. With two-dimensional gels, more than 80 interphase species were found as components of metaphase chromosomes. The degree of conservation of HeLa nonhistone antigens with other chromatin sources was calculated through densitometry of autoradiograms and stained gels. Chromatin was obtained from chicken erythrocytes, Novikoff rat hepatoma cells, and mouse L cells, and the presence of one-quarter to one-third of the nonhistone antigens of HeLa chromatin was demonstrated.

Animals↗
PubMed1990

Identification of mitotic (CDC2) and interphase histone H1 kinases by nondenaturing gel electrophoresis and peptide assays.

A new method of nondenaturing gel electrophoresis and a specific peptide based assay were used to study the histone kinases in mitotic and interphase mouse fibroblasts. The gels resolved four activities, one of which was shown to be the mitotic (CDC2) H1 kinase by virtue of its antigenicity. A new peptide substrate for the CDC2 kinase was phosphorylated by both S-phase and mitotic cell extracts and reacted with two protein kinases in the gels. Since the interphase enzyme did not react with the antibody, the results suggest that it is either a "masked" form of CDC2 or a second enzyme, functionally related to CDC2, which is responsible for the interphase phosphorylation of H1.

Animals↗
PubMed1986

Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes.

The cytoplasmic factor responsible for chromosome condensation was introduced into mouse zygotes at different times after fertilization by fusion of the zygotes with metaphase I oocytes. In 72% of heterokaryons obtained after fusion of early zygotes (14-18 hr post-human chorionic gonadotrophin (HCG) with oocytes, the male and female pronuclei of the zygote decondensed. At the same time, the oocyte chromosomes became enclosed in a nuclear envelope and decondensed to an interphase state. However, in the rest of the heterokaryons, the chromatin of the pronuclei condensed to metaphase chromosomes, thus resulting in three sets of chromosomes. Fusion of zygotes that had begun DNA synthesis (20-22 hr post-HCG) with oocytes induced chromosome condensation of the pronuclei in 76% of the cases. In some heterokaryons, however, the oocyte chromosome decondensed to an interphase state similar to the zygote pronuclei. Fusion between late zygotes (27-29 hr post-HCG) with oocytes resulted in chromosome condensation of the pronuclei in all heterokaryons. On the basis of these results, the formation of the pronuclei and their progression toward mitosis in the zygote may be explained by changing levels of a metaphase factor in the cell, or by a balance between interphase and metaphase factors.

Animals↗
PubMed1984

Injected mitotic extracts induce condensation of interphase chromatin.

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.

Animals↗
PubMed1986

On the position of nucleolus organizer regions (NORs) in interphase nuclei. Studies with a new, non-autoradiographic in situ hybridization method.

The distribution of 18S and 28S ribosomal RNA (rRNA), i.e. the chromosomal nucleolus organizer regions (NORs) was visualized in interphases and metaphases of non-stimulated and phytohemagglutinin (PHA)-stimulated human lymphocytes with a recently developed non-autoradiographic in situ hybridization method. This procedure involves mercurated RNA as a probe and a sulfhydryl-trinitrophenyl-mercury binding ligand and FITC-labelled antibodies as detection system. Silver staining was used to visualize nucleoli in interphase. In the secondary constriction of all ten acrocentric chromosomes, varying amounts of rDNA were detected. In the interphase nuclei of most of the non-stimulated human lymphocytes, only one small nucleolus could be seen. The in situ hybridization, however, revealed several agglomerations of rDNA scattered over the whole nuclear area, clearly outnumbering the number of nucleoli in these cells. This means that not all of the NORs are transcriptionally active in non-stimulated lymphocytes and that these inactive NORs lie at a distinct distance from the active ones. With PHA stimulation (transforming the small lymphocytes from peripheral blood into large, lymphoblast-like cells) the number of nucleoli increased slightly, whereas the number of separable rDNA spots decreased. This means that in the course of PHA-induced cellular activation, formerly inactive NORs become transcriptionally active and tend to associate with one another. This indicates the occurrence of movements of the NORs within the nucleus, depending on their transcriptional activity.

Adult↗
PubMed1991

Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells.

Among a library of monoclonal antibodies (mAbs) recognizing developmental markers in the chick embryo, mAb CC-3 was selected because of its differential immunostaining of mitotic cells. The intracellular distribution of the CC-3 antigen (CC-3a) throughout the cell cycle was visualized by immunolocalization. In interphase cells CC-3a resided in the nucleus and was arranged in distinct extranucleolar clusters. At prophase, the nuclear reactivity of CC-3a considerably increased and subsequently extended to the cytoplasm at metaphase. From metaphase through anaphase, most of the reactivity was associated with the mitotic apparatus. During cytokinesis CC-3a was detected in the mid-body and also in discrete speckles dispersed throughout the cytoplasm. The initial interphase pattern was then restored in the two daughter nuclei. Immunoblot analysis demonstrated that a 255-kDa phosphoprotein was present only in the interphase nucleus and that a complete new set of phosphoproteins accounted for the mitotic cell reactivity. The binding of CC-3 was dependent on the phosphorylation of its antigens. CC-3a is an evolutionary conserved molecule; it is present in such phylogenetically distant species as Drosophila and humans. Furthermore, the unique behavior of CC-3 on sections of normal, embryonic, and regenerative tissue and in cell culture immunostaining make it a reliable tool to identify mitotic foci.

Animals↗
PubMed1984

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes.

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.

Antigens↗
PubMed1985

ADP-ribosylation of metaphase and interphase nonhistones using [3H]adenosine as a radioactive label.

ADP-ribosylation of HeLa nonhistone proteins was investigated by using [3H]adenosine as an in vivo radioactive label. The aim was to determine basic differences in the patterns of modification of interphase and metaphase nonhistones. Fluorography revealed a relatively small number of modified proteins for isolated metaphase chromosomes. In addition to the core histones, a protein of 116 kDa, which is identified as poly-(ADP-ribose) polymerase, was a primary acceptor of [3H]adenosine. Two-dimensional gels revealed a profound difference in the modification of metaphase and interphase nonhistones. For interphase nuclei, 3H label was distributed among a large number of nonhistone acceptors.

Adenosine↗
PubMed1990

Changes in frequency and localization of human X- and Y-chromatin bodies at interphase during in vitro cellular aging.

We have investigated the degree of hypodiploidy of human X (inactive) and Y chromosomes and their relative localization in the interphase nuclei during in vitro aging of diploid fibroblasts. It is found that significant proportions of both female and male cells lose the inactive X and Y chromosome, respectively during cellular aging. Our results from fibroblasts are consistent with comparable findings of other investigators utilizing lymphocytes and bone marrow cells. However, we have observed a relatively higher proportion of X and Y chromosomal hypodiploidy in older fibroblasts than the frequencies reported for lymphocytes or bone marrow cells from aged people. Significant changes in the relative localization pattern of the inactive X and Y chromosomes in the nuclei are also noted during in vitro aging of female and male cells, respectively, and these changes in localization pattern are not identical in both sexes. We believe that, during cellular aging, the analysis of sex chromosomal aneuploidy at interphase is highly likely to provide more accurate results as opposed to the analysis of metaphase chromosomes since the latter is dependent upon the divisional capacity of cells which declines with age. Analysis of interphase cells also avoids the artifacts that accompany metaphase chromosome preparations.

Aging↗
PubMed1994

Detection of i(17q) chromosome by fluorescent in situ hybridization (FISH) with interphase nuclei in medulloblastoma.

Medulloblastomas are the most frequent primitive neurectodermal tumors in children. An isochromosome for the long arm of 17, i(17q), is found in 30% of medulloblastomas. For some authors, this abnormality is observed in cases with a shorter survival time. In our cytogenetic studies of 30 medulloblastomas, we observed i(17q) in only three cases, a monosomy 17 in two cases, a monosomy 22 in four cases, nonspecific numerical or structural abnormalities in five cases, and normal karyotypes in 12 cases. We compared the results of karyotypic analysis after culture and FISH with a chromosome 17 alpha satellite DNA probe on interphase nuclei in five cases of medulloblastoma. In one case, i(17q) was only observed in four cells in karyotypic analysis, in three cases a normal karyotype was found, and in one case karyotypic analysis was impossible. In all of these cases, i(17q) was observed in a great number of nuclei by FISH on interphase nuclei. Our study shows that the FISH on interphase nuclei permitted us to observe i(17q) in the cases where it was not or could not be completely detected by karyotypic analysis. The association of these two techniques is required to detect i(17q), an abnormality whose prognosis value in medulloblastomas is now recognized.

Adolescent↗
PubMed1995

Advantages and limitations of using fluorescence in situ hybridization for the detection of aneuploidy in interphase human cells.

Fluorescence in situ hybridization with chromosome-specific DNA probes is being increasingly utilized for the detection of chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Although potentially a powerful technique, FISH studies for aneuploidy can be heavily influenced by cellular phenomena and hybridization artifacts which make the performance and interpretation of the results difficult. As a consequence, frequently hyperdiploid frequencies are reported in the literature which are substantially higher than one would expect based upon frequencies seen in conventional metaphase analyses. In this article, a number of the potential pitfalls that we have encountered while performing FISH analyses for aneuploidy are discussed and their potential impact on the observed hybridization frequencies is described. After considering these factors, the frequencies of lymphocyte nuclei containing 3 and 4 chromosome copies are compared between metaphase values obtained from published human population studies and interphase values obtained from similar studies using FISH. It is concluded that by using caution in the evaluation of slides, interphase studies using FISH to detect hyperdiploidy and polyploidy can provide estimates of numerical alterations which closely reflect those seen during metaphase analysis using either FISH or conventional approaches. However, due to the inability of interphase analysis to distinguish hyperdiploidy from polyploidy as well as other potential problems, frequencies of aneuploid nuclei obtained using single label FISH should only be considered approximations of absolute frequencies. For additional accuracy, multi-color FISH with two or more different probes should be performed.

Aneuploidy↗
PubMed1985

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. III. Transition between mitotic/cytokinetic and interphase microtubule arrays.

Immunofluorescence microscopy of flowering plant root cells indicates that the earliest interphase microtubules appear during cytokinesis, radiating from the former spindle poles and subsequently from the nuclear envelope. They form networks that have microtubule focal points in the cortex underlying cell faces and in the cytoplasm between the nucleus and cortex. Cortical networks are rapidly replaced by the highly aligned array normally associated with interphase. An antibody that in animal cells identifies the location of pericentriolar material, the site of microtubule initiation, is also localized around the plant cell nuclear envelope at the time that putative early interphase microtubule networks are seen.

Cell Cycle↗
PubMed1992

Effects of 5'-iododeoxyuridine on the repair of radiation induced potentially lethal damage interphase chromatin breaks and DNA double strand breaks in Chinese hamster ovary cells.

The effect of 5'-iododeoxyuridine incorporation into DNA on radiation sensitivity, cellular repair capability, induction and repair of interphase chromatin breaks, as well as induction and repair of DNA double strand breaks was investigated in plateau-phase Chinese hamster ovary cells exposed to X rays. Repair of potentially lethal damage, as measured by delayed plating plateau-phase cells, was used to assay cellular repair capacity. Induction and repair of interphase chromatin breaks were assayed by means of premature chromosome condensation, whereas induction and repair of DNA double strand breaks were assayed by pulsed field gel electrophoresis. Incorporation of 5'-iododeoxyuridine into DNA sensitized cells to radiation. Radiosensitization increased with increasing percent thymidine replacement and was accompanied by an increase in the number of chromatin breaks scored per Gy and a small increase in the number of DNA double strand breaks produced. Cells grown in the presence of 5'-iododeoxyuridine were able to repair potentially lethal damage. When the comparison was made at equal doses, the extent of this repair was higher but its rate slower in 5'-iododeoxyuridine containing cells. At equal survival levels, cells that had incorporated IdU also repaired PLD to a slightly higher extent than control cells grown in IdU free medium. The magnitude of potentially lethal damage repair increased as cells "aged" in the plateau-phase, particularly for high 5'-iododeoxyuridine concentrations (8 microM). Incorporation of 5'-iododeoxyuridine reduced the rate of repair of interphase chromatin breaks and the rate of repair of DNA double strand breaks (both the fast and the slow component). The results suggest that reduction in the efficiency of repair of DNA double strand breaks and chromatin breaks, produced by radiation in 5'-iododeoxyuridine containing cells, is one determinant of the radiosensitization observed.

Animals↗
PubMed1993

Determination of the radiation sensitivity of the stromal cells in the murine long-term bone marrow culture by measuring the induction and rejoining of interphase chromosome breaks.

PURPOSE: To determine the radiosensitivity of bone marrow stromal cells, the rate of interphase chromosome breakage and rejoining of stromal cells in the murine long term bone marrow culture and of human skin fibroblasts were compared. METHODS AND MATERIALS: The cells were irradiated with doses up to 6 Gy and repair times up to 6 hr were investigated. After induction of premature chromosome condensation by fusing the cells with mitotic HeLa cells, the number of interphase chromosome fragments was counted. RESULTS: The number of radiation induced breaks was found to be not significantly different for both cell types with 6.16 +/- 0.26 breaks per Gray for the fibroblasts and 5.96 +/- 0.20 breaks per Gray for the stromal cells. A significant difference was observed in the repair rate. The fibroblasts rejoined 39.6% of the breaks induced initially during the first hour after irradiation and 5.6 +/- 1.84 breaks remained unrejoined after 6 hr, while the stromal cells were able to rejoin 63.2% in 1 hr and had 2.05 +/- 0.07 breaks unrejoined after 6 hr. CONCLUSION: If the well substantiated assumption is made, that the capacity to repair DNA double strand breaks or interphase chromosome breaks is correlated with the cellular radiosensitivity, this finding indicate, that murine bone marrow stromal cells are more radioresistant than human skin fibroblasts.

Animals↗
PubMed1993

Increased frequency of formation of interphase ring-chromosomes in radiosensitive irs-1 cells exposed to X-rays.

Induction and rejoining of interphase chromosome breaks were measured, after exposure to X-rays, in plateau-phase Chinese hamster V79 cells and in a radiosensitive mutant cell line derived from them, iris-1, using premature chromosome condensation (PCC). There was no difference in the induction of interphase chromosome breaks per Gy between the radiosensitive mutant cells and wild-type V79 cells despite the large differences in their radiosensitivity; an induction of 2.85 +/- 0.05 breaks/cell/Gy was measured in both cell lines. Also, rejoining of interphase chromosome breaks proceeded in the two cell lines with similar kinetics (t1/2 = 26.2 min). In contrast, ring chromosome formation was higher in irs-1 cells, as compared to wild-type V79 cells (0.78 versus 0.44 after 6 h of repair). These results confirm previous observations suggesting that a general deficiency in the rejoining of DNA dsb is unlikely to be a direct cause of the increased radiosensitivity of irs-1 cells, and are consistent with the hypothesis that the increased radiosensitivity of these cells derives from an increase in the probability of misrepair.

Animals↗
PubMed1999

p42-MAP kinase is activated in EGF-stimulated interphase but not in metaphase-arrested HeLa cells.

It is known that cellular signals produced in response to an inappropriate spindle formation cause the cell to be arrested at metaphase (M) in the cell cycle. We report here that the 42-kDa isoform of MAPK (ERK2) was tyrosyl-phosphorylated and activated in response to epidermal growth factor (EGF) in interphase but not in M-arrested HeLa cells. However, the basal level of activity of M-arrested cells was higher than that of interphase, although the overall tyrosyl phosphorylation content was small. Further, the EGF receptor and its associated proteins GTPase-activating protein and phospholipase C were phosphorylated in M-arrested cells to a lower extent than they were in interphase. This implies that in spite of its high level of basal activity, the scarcity of MAPK activation in mitosis in response to EGF stems from an early impairment of phosphorylation of the receptor and neighboring proteins. The biological significance of these results underlies the importance of keeping the cell sheltered from extracellular signals when it undergoes division.

Enzyme Activation↗