Human Lymphocyte Nuclei Research Articles

Three-dimensional distribution of centromeric or paracentromeric heterochromatin of chromosomes 1, 7, 15 and 17 in human lymphocyte nuclei studied with light microscopic axial tomography

Light microscopic axial tomography was applied to examine the position of peri- or paracentromeric chromosomal targets in nuclei of PHA-stimulated human lymphocytes following two color fluorescence in situ hybridization with DNA probes for 1q12 and 15p or 7c and 17c. Evaluation of hybridized nuclei was performed in a glass capillary device. By turning the capillary around its longitudinal axis each nucleus could be viewed at any desired angle. To measure the true three-dimensional distance between two chromosomal targets, the nucleus was turned around until both targets were positioned in the same focal plane. Similarly, the true distance between a target region and the center of the nucleus was estimated. The results provide evidence for differences in the three-dimensional nuclear distribution of the target regions. In particular, 7c was positioned more peripherally than the other chromosomal targets. Experimental data were compared with several models for a distribution of chromosomal targets under topological constraints. These models take into account spatial limitations of the distribution of the chromosome territories which harbor the hybridized targets.

Three‐dimensional distribution of centromeric or paracentromeric heterochromatin of chromosomes 1, 7, 15 and 17 in human lymphocyte nuclei studied with light microscopic axial tomography

Three‐dimensional distribution of centromeric or paracentromeric heterochromatin of chromosomes 1, 7, 15 and 17 in human lymphocyte nuclei studied with light microscopic axial tomography

Simultaneous detection of X-chromosome loss and non-disjunction in cytokinesis-blocked human lymphocytes by in situ hybridization with a centromeric DNA probe; implications for the human lymphocyte in vitro micronucleus assay using cytochalasin B.

A methodology for the simultaneous detection of chromosome loss and gain in mammalian cells has been developed which is based upon the analysis of chromosome distribution in daughter nuclei of binucleated human lymphocytes. X-chromosome distribution was followed by in situ hybridization, using a commercial biotinylated DNA probe specific for the centromeric alphoid sequences of human X-chromosome. In order to optimize the experimental protocol for the use of cytokinesis-blocked lymphocytes in aneuploidy assays, the effect of harvest time and cytochalasin B (Cyt B) dosage upon chromosome distribution was investigated. To this end, lymphocyte cultures were treated 44 h after mitogen stimulation with different dosages of Cyt B and collected at 60, 66 and 72 h. High rates of binucleated cells with unbalanced chromosome distribution (two spots in one nucleus and none in the other in male cells; three spots in one nucleus and one in the other in female cells) and abnormal spot number (more than or less than two per male cell or four per female cell) were observed at 66 and 72 h in cultures treated with the lowest Cyt B dose (3 micrograms/ml). In contrast, low frequencies of unbalanced or abnormal binucleated cells were observed at 60 h with both 3 and 6 micrograms/ml Cyt B. These results indicate that binucleated lymphocytes with low background frequencies of malsegregation (required for the analysis of induced aneuploidy), can be obtained by harvesting lymphocyte cultures 60 h after stimulation (16 h after Cyt B block).(ABSTRACT TRUNCATED AT 250 WORDS)

A nuclear protein with enhanced binding to methylated Sp1 sites in the AIDS virus promoter.

We report here the discovery of HMBP, a protein in nuclei of human T-helper lymphocytes and other human cell types, which binds with enhanced affinity to a promoter element in the HIV-1 long terminal repeat when that element is methylated at CpGs, the target site of the human DNA methyltransferase. This promoter element contains three (degenerate) binding sites for Sp1, a general activator of transcription. Gel shift assays and footprinting experiments indicate that HMBP binding overlaps two of these methylated Sp1 sites. Although HMBP binds these methylated Sp1 sites, it does not bind consensus Sp1 sites. Competition studies, differences in binding site specificities, binding conditions, and, in some cases, chromatographic separation further distinguish HMBP from Sp1 and from each of four previously identified methylated-DNA binding proteins. HMBP binds hemimethylated DNA in a strand dependent manner. These binding characteristics suggest that HMBP may recognize newly replicated DNA and thereby play a role in differentiation. If HMBP is able to compete with Sp1 for binding at methylated, non-consensus Sp1 sites in vivo and repress transcription, it may play a role in AIDS latency.

Automated detection of radiation-induced chromosome aberrations following fluorescence in situ hybridization

The cytogenetic detection of rare chromosome aberrations induced by ionizing radiation requires the evaluation of large numbers of cells. In this report, an image analysis program based on thresholding of grey level histograms is described for a rapid automated detection of chromosome translocations in metaphase spreads from human lymphocytes following irradiation and chromosome in situ suppression (CISS) fluorescence in situ hybridization (FISH). To classify a metaphase spread as “normal”, “aberrant”, or “excluded”, a minimum medium time of less than two seconds was required on a general purpose Personal Computer. Using appropriately stained specimen, for the false classification rate an upper limit of about 10% was estimated. The upper limit for the rate of “excluded” cells was estimated to be about 11% (99% confidence ranges). FISH-procedures allow to score chromosome aberrations also in the interphase nucleus. To apply simple threshold algorithms for segmentation of the FISH-stained nuclear areas, it is required that the grey level contrast is sufficiently high. The preliminary results presented here on the image analysis of human lymphocyte nuclei suggest the feasibility of such an approach.

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.

Automated detection of radiation-induced chromosome aberrations following fluorescence in situ hybridization.

The cytogenetic detection of rare chromosome aberrations induced by ionizing radiation requires the evaluation of large numbers of cells. In this report, an image analysis program based on thresholding of grey level histograms is described for a rapid automated detection of chromosome translocations in metaphase spreads from human lymphocytes following irradiation and chromosome in situ suppression (CISS) fluorescence in situ hybridization (FISH). To classify a metaphase spread as "normal", "aberrant", or "excluded", a minimum medium time of less than two seconds was required on a general purpose Personal Computer. Using appropriately stained specimen, for the false classification rate an upper limit of about 10% was estimated. The upper limit for the rate of "excluded" cells was estimated to be about 11% (99% confidence ranges). FISH-procedures allow to score chromosome aberrations also in the interphase nucleus. To apply simple threshold algorithms for segmentation of the FISH-stained nuclear areas, it is required that the grey level contrast is sufficiently high. The preliminary results presented here on the image analysis of human lymphocyte nuclei suggest the feasibility of such an approach.

Targeted cytogenetic analysis of gastric tumors byin situhybridization with a set of chromosome-specific dna probes

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of ten gastric adenocarcinomas. All tumors (eight male, two female patients) were histologically moderately or poorly differentiated and nine of ten had metastasized to regional lymph nodes. The authors applied a set of satellite DNA probes, specific for chromosomes 1, 7, 17, X, and Y in order to detect numerical chromosome aberrations in freshly isolated tumor cell nuclei. Normal diploid human lymphocyte nuclei and, in a number of cases, normal gastric mucosa served as controls. Parallel with the hybridization experiments DNA flow cytometric study of acridine orange (AO)-stained tumor cells was carried out. By means of FISH the authors found seven cases to be aneuploid, the other three cases appeared diploid. This was confirmed by DNA flow cytometric analysis with AO. Furthermore, loss of the Y chromosome in a high percentage of cells was seen by FISH in six of eight tumors from male patients. In the other two male samples a possible loss was observed in a small proportion of cells (15%). In three patients from whom the authors had normal gastric mucosa the Y loss was restricted to the tumor cells. These data indicate that in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors, at least with respect to the detection of numerical chromosome abnormalities.

Changes in Ca,Mg-dependent DNA endonuclease in isolated human lymphocyte nuclei in lymphoproliferative diseases

Changes in Ca,Mg-dependent DNA endonuclease in isolated human lymphocyte nuclei in lymphoproliferative diseases

Cytofluorometric determination of protein‐bound thiols and DNA in cell nuclei

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non-proliferating lymphocytes distinct DNA- and PSH-values could be measured. The PSH-values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S-phase and was almost doubled during the cell generation cycle from G1- to G2-phase. Cell line and cell cycle-dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.

Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy

A fluorescent in situ hybridization procedure with a chromosome 1-specific (1q12) repetitive satellite DNA probe was used to label the 1q12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The 1q12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.

Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Nuclear lysate sedimentation measurements of peripheral blood lymphocytes from radiotherapy patients.

When isolated nuclei of human lymphocytes are challenged with 2 M NaCl a histone-free-DNA-protein (HF-DNA) complex is released. In a linear sucrose gradient (pH 8.0) the sedimentation distance of HF-DNA is reduced when immediately isolated from cells irradiated in vitro. At low doses, if irradiated cells are incubated at 37 degrees C the sedimentation behaviour approaches that of unirradiated cells (i.e. repair). In the present study such repair was usually complete within 1 h. The radiation damage to lymphocytes from a healthy donor group and three patient groups consisting of new patients (before radiotherapy), well patients (2 to 6 years post-radiotherapy) and patients with complications attributed to radiotherapy was similar. The lymphocytes from most healthy donors and new patients demonstrated complete repair of radiation damage following an incubation of 1 h at 37 degrees C. However, 2/29 (6.9 per cent) healthy donors and 2/25 (8 per cent) new patients demonstrated poor repair. Of those patients now attending with 'bowel complications' attributed to radiotherapy 7/16 (44 per cent) demonstrated poor repair. In contrast, all those (11/11) described as 'well and complication-free' showed good repair.

Characterization of thyroid hormone receptors in human IM-9 lymphocytes.

Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37 degrees C, nuclear binding of [125I]T3 was saturable (Kd 34 +/- 6 pmol/l) and of finite capacity (approximately equal to 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 +/- 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 +/- 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3'-isopropylthyronine, 89; T4, 32; 3,5-dimethyl-3'isopropylthyronine 2; 3,5-T2, 0.7, rT3, 0.4; 3'5'-T2, less than 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

A bicistronic Epstein-Barr virus mRNA encodes two nuclear proteins in latently infected, growth-transformed lymphocytes.

EBNA2 is a nuclear protein expressed in all cells latently infected with and growth transformed by Epstein-Barr virus (EBV) infection (K. Hennessy and E. Kieff, Science 227:1230-1240, 1985). The nucleotide sequence of the EBNA2 mRNA (J. Sample, M. Hummel, D. Braun, M. Birkenbach, and E. Kieff, Proc. Natl. Acad. Sci. USA 83:5096-5100, 1986) revealed that it begins with a 924-base open reading frame that has an unusual potential translational initiation site (CAAATGG). This open reading frame is followed by 138 nucleotides with only one highly unlikely translational initiation site (TACATGC), which would translate a pentapeptide before the next stop codon. The last part of the mRNA is the open reading frame which encodes EBNA2. In this paper, we demonstrate that the 924-base open reading frame translates a 40-kilodalton protein in vitro or in murine cells transfected with the EBNA2 cDNA under control of the murine leukemia virus long terminal repeat. A protein of identical size was detected in EBV-transformed, latently infected human lymphocyte nuclei by using antibody specific for the leader open reading frame expressed in bacteria. Therefore, this is a rare example of a mRNA which translates two proteins from nonoverlapping open reading frames. Since the protein encoded by the leader of the EBNA mRNA is expressed in all nuclei of a latently infected cell line, it was designated EBNA-LP. EBNA-LP localizes to small intranuclear particles and differs in this respect from EBNA1, EBNA2, or EBNA3. EBNA-LP is not expressed in an EBV-transformed marmoset lymphocyte cell (B95-8) or in one EBV-infected Burkitt tumor cell line (Raji) but is expressed in three other Burkitt tumor cell lines (Namalwa, P3HR-1, and Daudi).

Complementary replica of freeze-fractured human lymphocyte nuclei.

Complementary replicas of freeze-fractured human lymphocyte nuclei were obtained, and were studied with the electron microscope. Complementarity was evident in cross-fractured nuclear pore complexes and in particle-free membrane areas. Explanations for the observed structural peculiarities are considered.

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes.

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter----Xp22.2::Yq11----Yqter) contained in the Chinese hamster X human hybrid cell line 445 X 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.

The intrinsic substrate specificity of a cyclic nucleotide-independent protein (histone) kinase

Phosphorylation and dephosphorylation of proteins play a pivotal role in the regulation of intracellular processes (reviewed in [l-3]). Beside several well-characterized protein kinases a number of protein phosphorylating enzymes have been found; however, their natural substrates and regulatory properties are not known. A possible way to identify these enzymes is the determination of their intrinsic substrate specificity; i.e., the most important residues of the amino acid sequence that they recognize in their substrate. A cyclic nucleotide-independent histone kinase (designated as HK II) was described in 1451. We have also reported a type of cyclic nucleotide-independent histone kinase found in the cytoplasm and nucleus of human tonsillar lymphocytes [6,7] and in the extract of bovine thymus [8]. This enzyme phosphorylates the Ser-32 residue of calf thymus H2b histone and a synthetic peptide containing the amino acid sequence of H2b from Gly-26-Lys-34, but it does not act on Ser-36 which is phosphorylated preferentially by the catalytic subunit of the cyclic AMP-dependent protein kinase 19-l 11. Separation of the cyclic nucleotide-independent histone kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase was carried out by DEAE-cellulose chromatography in the presence of cyclic AMP, as in 181. The histone kinase fraction obtained by this procedure from bovine thymus was dialyzed against 0.005 M potassium phosphate (pH 7.5) and it was applied onto a hydroxylapatite (Bio-Gel HTP) column. The column was washed by 5 vol. of each of 0.01 M, 0.07 M, 0.085 M, 0.1 M and 0.4 M potassium phosphate (pH 7.5). The histone kinase was eluted by the last volumes of 0.07 M and by the first volume of 0.085 M potassium phosphate. The specific activity of the enzyme preparation obtained was lo-12 nmol phosphate transferred mg protein -’ . min -‘, as measured in the presence of 1 mg histone Hi/ml and 2 x lo-’ M ATP. This preparation yielded a single (Mr 55 000) histone kinase peak on Sephadex G-100 gel-chromatography.

The site of histone H2b phosphorylated by a cyclic nucleotide independent histone kinase

A cyelic nucleotide independent histone kinase was demonstrated in bovine thymus extract. This enzyme was very similar to that found previously in the cytoplasm and nucleus of human tonsillar lymphocytes (Faragó et al., 1973, Biochim. Biophys. Acta 297, 517 and 1974, ibid 370, 459). High-performance liquid chromatography of the tryptic phosphopeptides of calf thymus histones H1 and H2b phosphorylated by the histone kinase or by the catalytic subunit of cylic AMP dependent protein kinase showed that the intrinsic substrate specificity of these enzymes differed significantly. Under our experimental conditions the histone kinase phosphorylated preferentially the Ser-32 residue, and it did not phosphorylate the Ser-36 residue of histone H2b, while Ser-36 was phosphorylated preferentially by the cyclic AMP dependent protein kinase. A peptide containing the amino acid sequence of histone H2b from Gly-26 to Lys-34 (Gly-Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-Ala) was synthesized. This peptide was a competitive inhibitor of histone H1 phosphorylation by the histone kinase and it was also a substrate for this enzyme.

Position of chromosomes in the human interphase nucleus. An analysis of nonhomologous chromatid translocations in lymphocyte cultures after Trenimon treatment and from patients with Fanconi's anemia and Bloom's syndrome.

The problem of localization of chromosomes in relation to each other in the interphase nucleus of human lymphocytes was investigated by analysis of chromatid and chromosome aberrations observed in lymphocyte cultures of three patients with Fanconi's anemia, one patient with Bloom's syndrome, and in Trenimon-treated (Trenimon, Bayer) normal cells. Distribution of open gaps and breaks is highly correlated with chromosome length and distribution of breaks involved in chromatid translocations in Fanconi's anemia and in Trenimon-treated cells. Both correlations are much lower in Bloom's syndrome. In Fanconi's anemia and in normal cells after Trenimon-treatment, the majority of chromatid translocations are between nonhomologous chromosomes, whereas in Bloom's syndrome mainly homologous chromosomes are involved. Statistical localization of chromosomes in relation to each other in the three-dimensional space by multidimensional scaling gives results consistent with the limited amount of independent evidence.