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At least 793 records · Page 44Linked to original sources
PubMed1988

Relative deoxyribonucleic acid content of interphase leukocytes by flow cytometry: a method for indirect diagnosis of chromosomal abnormalities with potential for prenatal diagnosis.

A major goal of prenatal cytogenetic analysis is the development of minimally invasive techniques by which all pregnancies may be screened. That nucleated fetal cells exist in the maternal circulation raises the possibility that their cytogenetic status could be determined. However, these cells may not respond to commonly used mitogens. Thus it will probably be necessary to develop methods to analyze nondividing (interphase) cells. We therefore evaluated the use of flow cytometry as a means of determining whether the relative deoxyribonucleic acid content of G0-G1 leukocytes (expressed as the deoxyribonucleic acid index) could be used to verify aneuploidy or other chromosomal abnormalities. Our findings indicate this is indeed possible. We determined that the mean deoxyribonucleic acid index of circulating leukocytes from normal adult men (n = 15) was significantly different from that of leukocytes from normal adult women (n = 15). Similar results were obtained in leukocytes from umbilical cord blood of normal male neonates (n = 15) and normal female neonates (n = 15). Most importantly, values for leukocytes from each of 13 aneuploid individuals fell outside the range of values for leukocytes from normal adults of the same sex.

Adult↗
PubMed1991

c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes.

Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.

Animals↗
PubMed1988

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes.

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.

Cell Line↗
PubMed1989

Distribution of c-myc, c-myb, and Ki-67 antigens in interphase and mitotic human cells evidenced by immunofluorescence staining technique.

Using specific antibodies and the immunofluorescence staining technique we found a similar subcellular distribution pattern of the cellular proto-oncogene proteins c-myc and c-myb in interphase and mitotic HL60 and Molt4 cells. Antibodies against c-myc as well as those against c-myb protein gave rise to a nuclear staining excluding the nucleoli. In mitotic cells both proteins are apparently not associated with the chromatin of the condensed chromosomes, but appear diffusely distributed throughout the cytoplasm. In contrast, immunostaining using the proliferation marker antibody Ki-67 yielded in both cell lines several prominent specks in the nucleus and a weak finely dispersed staining throughout the nucleoplasm. No fluorescence was detectable in the cytoplasm. In dividing cells Ki-67 immunofluorescence was found to be associated with the surface of the chromosomes. The functional significance of the different localizations of the proteins is discussed in light of what is currently known about nuclear antigens.

Antigens, Surface↗
PubMed1989

VP-16-induced nucleotide pool changes and poly(ADP-ribose) synthesis: the role of VP-16 in interphase death.

Exposure of human promyelocytic leukemia cell line (HL-60) to VP-16 resulted in accumulation of DNA strand breaks. Concomitantly, intracellular NAD levels fell at 1 h, followed by declines in ATP at 2 h and in GTP, CTP, and UTP at 3 h. Furthermore, marked morphological changes, such as loss of microvilli or bleb formation, appeared at 4 h and cell death by 8-10 h. The addition of an inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide (5 mM), theophylline (2 mM), or thymidine (1 mM), prevented these sequential reductions of nucleotide pools and cell death. In fact, the activation of poly(ADP-ribose) synthesis was detectable within a few hours after treatment with VP-16, although it was smaller than that induced by N-methyl-N'-nitro-N-nitrosoguanidine. These results may suggest the possible role of activation of poly(ADP-ribosyl)ation in VP-16-induced nucleotide pool changes and subsequent interphase death.

Benzamides↗
PubMed1989

Ultrastructural changes in nucleoli and fibrillar centers under the effect of local ultraviolet microbeam irradiation of interphase culture cells.

As shown previously, ultraviolet (uv) microbeam irradiation of one of the two mature nucleoli within an interphase cell nucleus causes significant diminution and inactivation of the irradiated nucleolus and compensatory growth and activation of the nonirradiated one. In the present work we describe the results of an ultrastructural study of this phenomenon. The changes in the nucleoli were examined by means of complete series of ultrathin sections obtained from seven irradiated pig kidney cells. The compensatory hypertrophy of the nonirradiated nucleoli is shown to be accompanied by a nearly twofold increase in the number of fibrillar centers (FCs) and by a decrease in their linear dimensions compared with the control cells of the same ploidy. In the degraded nucleoli the number of FCs decreases, but their dimensions increase. Ultraviolet microbeam irradiation causes dramatic diminution of the dense fibrillar component within the irradiated nucleoli as well. The nucleolar capacity for compensatory hypertrophy indicates that in addition to active ribosomal genes, mature nucleoli also contain "silent" genes capable of being activated under extreme conditions to sustain the required level of rRNA synthesis. It is assumed that activation of latent ribosomal genes is accompanied by FC "fragmentation" without a considerable increase in their total volume per cell.

Animals↗
PubMed1989

The silver-stained proteins of interphasic nucleolar organizer regions as a parameter of cell duplication rate.

The relationship between interphasic silver-stained proteins of the nucleolar organizer regions (Ag-NOR proteins) and cell replication rate has been studied in 13 established neuroblastoma cell lines. The quantity of Ag-NOR proteins was measured in silver-stained cells by means of an automated image analyzer. The results indicated that the amount of Ag-NOR proteins is strictly proportional to the proliferative activity of the cells. Cell lines with a difference of only 4 h in doubling time were characterized by a statistically significant difference in Ag-NOR protein amount. The Ag-NOR protein quantity can therefore be used as a parameter for evaluating the cell proliferation rate.

Cell Division↗
PubMed1991

The effect of inducing agents on the numbers of interphase fibrillar centers in the U937 promonocytic cell line.

The mean numbers of interphase fibrillar centers have been determined in triplicate experiments, using the argyrophil (AgNOR) method. Promonocytic U937 cells were incubated with each of three inducing agents, namely, interleukin-6, granulocyte-macrophage colony-stimulating factor, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. The cells were examined at 24, 48, and 72 h during the induction periods and their doubling time and mean AgNOR score were calculated. After 72 h, the cells were maintained in culture for a further 24 h in the absence of an inducing agent and these parameters were determined again. It was found that whereas the unstimulated U937 cells had a mean value in the region of 50 AgNORs per nucleus, this diminished to about 20 after a 72 h incubation, but rose to 30 or more when the inducing agents had been withdrawn for 24 h. These observation confirm the results of previous studies using melanoma and HL60 cell lines: however, it has now been demonstrated that a variety of agents can modulate the numbers of fibrillar centers in a very similar way in a single cell line. Furthermore, we have shown that the "undifferentiated" U937 cell AgNOR score recovers when the agents no longer act upon the cells; this implies that fibrillar center numbers are intimately related to differentiation state in cell lines, as in the case in, for example, tumor specimens.

Cell Count↗
PubMed1992

Spatial rearrangement and enhanced clustering of kinetochores in interphase nuclei of dorsal root ganglion neurons in vitro: association with nucleolar fusion.

Interphase nuclei of several cell types display distinct, nonrandom arrangements of specific chromatin domains. It has been suggested that this arrangement is associated with the functional commitment of the cell and results from compartmentalization of specific DNA sequences to transcriptionally competent sites. In a test of the hypothesis that such topological organization is established during cellular differentiation, the spatial distribution of centromeres was determined, in dorsal root ganglion neurons in vitro, using immunocytochemistry in conjunction with fluorescence microscopy, confocal laser microscopy, and ultrastructural immunogold techniques. Kinetochores occurred as clusters, in association with nucleoli and with the nuclear envelope. Neurons at different stages of differentiation, as determined by nucleolar distribution, exhibited a distinct, stage-specific, spatial organization of kinetochores. Morphometric analyses, together with serial reconstruction, indicated that progressive clustering of kinetochores accompanies differentiation and that such clustering occurs in association with nucleolar fusion. The data presented indicate that the chromatin organization observed in the fully differentiated state may be the result of controlled rearrangements of specific chromatin domains during differentiation and that the mechanism governing such rearrangement and the process of cellular differentiation may be linked.

Animals↗
PubMed1992

Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes.

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

Amino Acid Sequence↗
PubMed1992

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.

Acetylation↗
PubMed1993

Combination of classical and interphase cytogenetics to investigate the biology of myeloid disorders: detection of masked monosomy 7 in AML.

Numerical abnormalities of chromosome 7 were detected by using fluorescence labeled in situ hybridization (FISH) procedure with a centromere-specific probe in four cases of acute myeloid leukemia (AML) and three cases of myelodysplastic syndromes (MDS). Comparison of these results with classical cytogenetic (CC) data demonstrated a good correlation between the two methods. FISH confirmed the finding of monosomy 7 in all patients who demonstrated this abnormality by CC. Two AML patients who did not show monosomy 7 by CC were unexpectedly found to contain this abnormality in 39.8% and 17% cells when examined by FISH. Given that our modified FISH method consistently yielded > 96% hybridization efficiency, these findings constitute an unexpected but real presence of monosomy 7 in a substantial number of interphase cells that had remained undetected by classical karyotyping. Finally, a number of maturing myeloid cells including granulocytes also demonstrated monosomy 7 by FISH, thereby confirming the ability of malignant cells to undergo differentiation. We conclude that FISH constitutes a highly sophisticated molecular technique which can be extremely useful in select cases for detecting 'masked monosomy 7' as well as helping to determine the lineage of terminally mature cells in AML, thereby providing a handle on the effects of cytokines or chemotherapy on normal vs leukemic clones.

Chromosomes, Human, Pair 7↗
PubMed1992

Uterine leiomyoma cytogenetics. III. Interphase cytogenetic analysis of karyotypically normal uterine leiomyoma excludes possibility of undetected trisomy 12.

Uterine leiomyoma, a benign tumor that histopathologically is rather homogeneous, was recently characterized cytogenetically. About 40% of the investigated tumors are associated with clonal chromosome abnormalities and five different subgroups have been identified, characterized by trisomy 12, t(12;14)(q14-15;q23-24), del(7q), t(1;2)(p36;p24), and 6p rearrangements. In our survey of 76 cases, trisomy 12 was observed in 10% of the abnormal cases. To exclude a possible underscoring of this abnormality, we reexamined 15 of the cases with normal karyotype by interphase cytogenetics using a chromosome 12 alphoid DNA probe.

Chromosomes, Human, Pair 12↗
PubMed1993

Heterogeneity in bladder cancer as detected by conventional chromosome analysis and interphase cytogenetics.

Thirty transitional cell carcinomas (TCCs) of the bladder were examined by classical chromosome counting to establish range, modal number, and percentage of metaphases with 2n, 3n, 4n, and > or = 5n chromosomes. In addition, fluorescence in situ hybridization (FISH) was applied to interphase nuclei to detect the percentage of tumor cells showing polyploidization and chromosome imbalance. In FISH, centromere-specific DNA probes for chromosomes 1, 7, 9, and 11 were used. The tumors were analyzed flow cytometrically to determine the DNA index (DI). Fourteen of 21 cases (67%) having a DI = 1 showed, after classical chromosome counting, in addition to a diploid model number, some cells with a 3n and 4n chromosome count. With FISH, eight cases (38%) showed a low percentage of cells with multiple signals for each of the probes, thus indicating polyploidization. In 13 (62%) cases, an imbalance between different chromosomes was detected. In nine tumors having a DI of 1.6 to 1.9, classical chromosome counting showed low percentages of > or = 5n cells in four cases, in addition to a triploid modal number. With FISH in six cases, a low percentage of cells showed five or more signals for each of the chromosomes, indicating polyploidization. In all cases, a chromosome imbalance was detected. With classical chromosome counting not all tumors can be analyzed. With FISH, small percentages of polyploid cells are not recognized. Both methods complement each other in that chromosome counting allows readier detection of heterogeneity in DNA-diploid tumors after polyploidization, whereas FISH allows efficient recognition of the chromosomes involved in the process of imbalance.

Adult↗
PubMed1994

Absence of trisomy 7 in nonneoplastic human ascitic and pleural fluid cells. An interphase cytogenetic study.

Trisomy 7 is a frequent aneuploid change in lymphomas, adenocarcinomas, and malignant mesenchymal and neurogenic tumors. Moreover, it has been observed in cultured and uncultured non-neoplastic cells from brain, kidney, liver, lung, and atherosclerotic plaques, among other tissues, opening debate on the role of this change in normal and neoplastic tissue. We used nonradioactive in situ hybridization (ISH) with a biotinylated chromosome 7-specific alpha-satellite DNA probe to seek an extra copy of chromosome 7 in ascitic and pleural fluid interphase cells from 26 donors. The donors comprised 24 patients with nonmalignant clinical history, one patient with non-Hodgkin's malignant lymphoma (positive control), and one patient with chronic myeloid leukemia (CML, negative control). The highest frequency of fluid cells with three hybridization signals in patients without neoplasia was 0.5%, in contrast to the frequency of 40.5% noted in the fluid cells of the patient with non-Hodgkin's malignant lymphoma. The results demonstrate that the frequency of trisomic cells in pleural as well as in ascitic fluid is very low, making possible use of the cells in ascitic or pleural fluids in identification of malignancy.

Adult↗
PubMed1994

Trisomy 12 in chronic lymphocytic leukemia. An interphase cytogenetic study by fluorescence in situ hybridization.

B-cell chronic lymphocytic leukemia (CLL) is a rare disorder in the Chinese population. We evaluated the use of fluorescence in situ hybridization (FISH) with a chromosome 12-specific probe in the detection of trisomy 12 in interphase cell of 19 Chinese CLL patients. FISH successfully detected trisomy 12 in two cases, one of which had normal conventional cytogenetic findings, giving an incidence of 10%. The low incidence of trisomy 12 in our CLL patients may also reflect a biologic difference of this rare disorder in our population, compared to that of the West.

Adult↗
PubMed1994

Interphase cytogenetics in paraffin-embedded sections from renal cortical neoplasms. Correlation with cytogenetic and flow cytometric DNA ploidy analyses.

Nonisotopic in situ hybridization (ISH) was performed on archival tissue sections from six renal cortical neoplasms and corresponding normal kidney tissue using pericentrometric (alpha-satellite) probes to chromosomes 1, 3, 7, 8, 10, 11, 12, 16, and 17. The results were correlated with classic cytogenetic analyses and flow cytometric DNA ploidy findings of these neoplasms. Our study shows that intratissue and intertissue ISH spot counts were generally homogeneous in normal kidney tissue and markedly heterogenous in tumor specimens. In all cases the correlation between the DNA Index and the ISH spot numbers was statistically significant. A correlation between modal and numerical chromosomal findings by cytogenetic analysis and ISH spot counts was found in three cases and was discordant in three cases. Our results also show that chromosomes 1, 3, 7, and 17 appear to manifest more propensity for aneuploidy than chromosomes 8, 10, 11, 12, and 16. We conclude that 1) numerical chromosomal aberrations by interphase in-situ hybridization correlate with DNA ploidy analysis in these tumors; 2) certain chromosomes may be more prone to aneuploidy than others; 3) determination of monosomy by ISH is less reliable on paraffin-embedded sections; and 4) the ISH technique complements conventional cytogenetic analysis in providing more information for the determination of cytogenetic aberrations and clonal heterogeneity in solid neoplasms.

Adenocarcinoma, Clear Cell↗
PubMed1990

Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei.

Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.

Adult↗