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Insights on diplochromosome structure and behaviour.

Diplochromosomes from colchicine-induced endoreduplicated HeLa cells were analyzed by light and electron microscopy. The patterns of C-, G-, NOR-, and late replicating DNA-rich bands obtained clearly indicate that the four chromatids constituting a diplochromosome are structurally identical and behave the same, at least in terms of replication kinetics and r-DNA transcription. Under the electron microscope, whole-mounted diplochromosomes appeared formed by two completely divided chromosomes showing no physical connection at their centromeric regions. The two chromosomes forming a diplochromosome seem to be held together by chromatin fiber loops connecting the two neighbouring chromatids of the sister chromosomes. These connecting loops clearly decrease in number as chromosome condensation progresses.

Bromodeoxyuridine↗

Replication variants of the human inactive X chromosome. II. Frequency and replication rate relative to the other chromosomes of the complement.

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25-27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.

Chromosome Aberrations↗

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells.

Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5',8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7--2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost negligible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.

Anemia, Aplastic↗

Diplotene chromosomes of Xenopus hybrid oocytes.

Observation on meiotic diplotene chromosomes from oocytes of Xenopus species and subspecies hybrids are reported. Species interrelationships are established on the basis of the number of bivalent in the respective hybrids. Polyploid oocytes were found and their origin by supplementary endoreduplication placed at the differentiation stage of gametogenesis when oogonia become oocytes. The significance of polyploid oocytes for speciation is discussed.

Animals↗

The structure and position of mouse centromeric heterochromatin in BrdU-labelled chromosomes and diplochromosomes stained by the pH 10.4 method.

A mouse cell line of C57Bl/6J spontaneous melanoma (clone PG 19), and a C-type virus transformed cell line (G-8 clone 124) originating from normal Balb/c mice were used in a study of the centromeric heterochromatin region of BrdU-labelled chromosomes stained by the Giemsa pH 10.4 method. Three possible explanations for the generation of compound lateral asymmetry within the centromeric heterochromatin region of the laboratory mouse are discussed: 1) inverted translocation; 2) centric fusion followed by paracentromeric fission and 3) inversion of part of the centromeric satellite DNA. These processes could be of considerable genetic and evolutionary significance. The non-random spatial position of unstained and dark stained C-bands in BrdU-labelled diplochromosomes of endoreduplicated cells can be explained as being due to the localization of the old and new DNA chains in a unineme chromatid model. The late replicating regions are shown to be located on the inside of the half-chromatid close to the axial symmetry axis of the metaphase chromosome.

Animals↗

Chemical induction of quadruple and octuple chromosomes in Chinese hamster CHO-K1 cells and relationship between their three-dimensional structure and spatial distribution of BrdU-labeled chromatids.

Double endoreduplication of Chinese hamster CHO-K1 cells that exhibited quadruple chromosomes at metaphase was induced by a combination of rotenone and ammonium vanadate treatments. Analysis of sister chromatid differential staining patterns (using 5-bromo-2'-deoxyuridine) revealed that approximately 50% of the quadruple chromosomes did not keep the scheme of "outside replication" of DNA. Based on the ratio of the staining patterns observed, we suggest that the two diplochromosomes forming a quadruple chromosome are held together by a physical link connecting the two original chromatids. Metaphases with octuple chromosomes were also produced by the same treatment. Each chromosome constituting an octuple chromosome was longer and thinner than ordinary metaphase chromosomes. This suggests incomplete chromosome condensation at metaphase. The majority of octuple chromosomes showed the eight constituent chromosomes to be so enmeshed that a planar alignment could not be observed in air-dried preparations.

Aneuploidy↗

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish, Poecilia formosa and its related, triploid unisexuals.

Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wide-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3-3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6-1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5-3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0-5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4-2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of 3H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.

Animals↗

Nuclear changes accompanying cell differentiation in stems of Pisum sativum L.

Nuclear DNA, nuclear protein and nuclear size have been measured in cells of the cortex, pith and vascular tissue from three successive internodes in the stem of Pisum sativum. New techniques of computer-linked cytophotometry were used to measure these parameters simultaneously in both section and squash preparations. In cortical cells no endoreduplicated nuclei were seen in the internodes measured. In cortical cells from the oldest internode measured, a population of large nuclei with the 2C DNA amount was observed which was not present in the younger internodes. In the oldest pith nuclei measured a few 8C nuclei were present, but maturing pith was most characterized by increasing nuclear size and the population of nuclei accumulating with the 4C DNA amount. Polyploid nuclei were present in all of the vascular tissue measured, including the youngest internode. Maturing vascular tissue was also characterized by increasing nuclear size. Nuclear protein measurements demonstrated a close link between nuclear protein and nuclear size and suggest that increased nuclear size, with constant DNA content, may be due to increased nuclear protein. This raises the question of the nature and function of this nuclear protein, perhaps more characteristic of differentiating cells than dividing cells.

Cell Differentiation↗

Human megakaryocytic progenitor cells.

Megakaryocytopoiesis represents one of several differentiation pathways that hematopoietic stem cells may enter. Cells representing intermediate stages of differentiation between pluripotent stem cells and maturing megakaryocytes are called megakaryocytic progenitor cells. They are identified in human bone marrow and peripheral blood by their ability to proliferate in culture (colony forming unit-megakaryocyte, CFU-M); at some point they lose the capacity for cell division and acquire the ability for endoreduplication of DNA, a phenomenon that is unique to the megakaryocyte lineage. This review summarizes current understanding of the biology of human megakaryocytic progenitor cells, including characterization of their proliferation potentials, their antigenic determinants, and the mechanisms that govern their proliferation and maturation. Finally the involvement of CFU-M in various disorders of thrombopoiesis is discussed.

Cell Differentiation↗

Proliferative activity of breast cancers increases in the course of genetic evolution as defined by cytogenetic analysis.

The prognostic value of proliferative activity and its relationship with steroid hormone receptors and histopathological grade have been demonstrated for breast cancers. However, nothing is known about the underlying mechanisms. In order to understand the chronology of the appearance of increased proliferative activities, we used a series of 760 consecutive breast cancers for which we had obtained S-phase fractions (SPFs) by DNA flow cytometry. When the absolute difference from a DNA index of 1.00 was compared to SPFs, a significant positive correlation was obtained (r = 0.39, p < 0.0001), indicating that the probability of observing a high SPF increases when tumors progressively deviate from diploidy. A highly significant correlation was observed for the hyperploid group when hypertetraploid tumors were excluded, as the SPFs increased progressively as the DNA indices decreased from 2.00 to 1.30. This observation suggested a relationship with the evolution of chromosomal abnormalities as determined by cytogenetic analysis. Indeed, in a subset of 52 cases for which sufficient metaphases were available, there was a highly significant correlation between the SPF values and the proportion of rearranged chromosomes in the tumor cells (r = 0.60, p < 0.0001). When SPFs were separated into low or high using the median value (4.5%), a correlation also existed with the genetic evolution, since they increased from diploidy to hypodiploidy and then, after endoreduplication, from tetraploidy towards triploidy, as determined by the chromosome counts. Our results substantiate the relationship between proliferative activity and steroid hormone receptors which follow the same model.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗

Multiple chromosomal associations and paracentromeric region instability in a case of acute leukemia.

A case of acute myelomonocytic leukema is described, which was characterized cytogenetically by the presence of centromeric elongations, somatic crossovers, selective endoreduplication figures, and multiple chromosomal clusters. The demonstration of these phenomena by selective staining techniques for the chromosome bands (Q, C, G and S) and the nucleolar areas (acridine-orange, amido black B 10) raises some biological aspects involved in the proliferation of leukemic cells, such as nucleolar persistance during the metaphase and the non-separation of chromatids in the clusters during the anaphase. These structural abnormalities may represent the background for the explanation of the appearance of subclones in neoplastic disorders.

Adult↗

Mechanisms of arsenic-induced cell transformation.

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Studies with cells in culture have provided some possible mechanisms by which arsenic and arsenical compounds may exert a carcinogenic activity. Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. The trivalent sodium arsenite was greater than tenfold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at nontoxic as well as toxic doses. At low doses, enhanced colony forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges, were induced by the arsenicals with similar dose responses. These results support a possible role for chromosomal changes in arsenic-induced transformation. The two arsenic salts also induced another form of mutation-gene amplification. Both sodium arsenite and sodium arsenate induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Developmental changes in the ploidy of mouse implanting trophoblast cells in vitro.

Shortly after the onset of implantation, polar mouse trophoblast cells proliferate and give rise to the ectoplacental cone, constituted by two distinct cell populations: undifferentiated, diploid cells and giant cells. Giant cells characteristically exhibit exaggerated dimensions and polyploid nuclei. In this study, we employ ectoplacental cones as a dynamic source of trophoblast giant cells to analyze cell proliferation, cell death, and ploidy under in vitro conditions. Our results show that DNA synthesis and the increase in the cell number are relevant only during the first 24 h of culture. Subsequently, DNA synthesis still occurs, mainly in the giant cell compartment, while the number of cells gradually decreases. Cell death by injury and apoptosis was also observed in the non-giant cell compartment of the ectoplacental cone. These findings suggest that the first 24 h of culture are crucial to the mitotic activity of the ectoplacental cone cells that gradually ceases, favoring the endoreduplication process. The DNA synthesis index during the subsequent experimental intervals emphasizes accumulation of DNA for the polyploidization. There was clear correlation between DNA content and nuclear dimension. The ploidy values for the trophoblast giant cells varied from 2C up to 368C in the giant cells, but were not as expressive as those known from in vivo conditions, probably due to the absence of regulatory factors specific to the embryonic-maternal interface. In situ hybridization and histochemistry for the nucleolus-organizing region showed that trophoblast nuclei have only two marker signals, indicative of a typical polytenic process. This present study elucidates important aspects of trophoblast behavior and provides new information on trophoblast physiology in vivo and in vitro.

Animals↗

Coordinates, DNA content and heterogeneity of cell nuclei and segments of the Caenorhabditis elegans intestine.

A specific protocol allowed to selectively stain and image the cell nuclei of the intestine of the nematode Caenorhabditis elegans. Digital processing of these images yielded quantitative information on the coordinates and the relative DNA content of these cell nuclei. With this technique, the exact locations (in relation to the length of worm or intestine) of the 20 intestinal cell nuclei of larval stage L1 and of the 34 intestinal cell nuclei of larval stages L2-4 and adults were determined. Tracking the DNA content of an individual intestinal cell nuclei allowed to study nuclear DNA endoreduplications during larval growth. The evaluation of the DNA content of all intestinal cell nuclei revealed a strong heterogeneity between the cell nuclei and intestinal segments with a maximum in segments int4-int5 in the L1 stage and int5-int6 in the L2-L4 stages. Minimum values were detected in the segments int2 and int8-int9 of all larval stages. Heterogeneity between the intestinal segments of C. elegans does not only concern the DNA content of cell nuclei, but also other cellular features like the quantity of intracellular vesicles or the absorption of particles out of the intestinal lumen.

Animals↗

Molecular characterization of the expression of distinct classes of cyclins during the early development of tomato fruit.

Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds from two distinct phases of growth, essentially cell division and cell expansion. In this study, we investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins, during tomato fruit development. We isolated six genes designated Lyces;CycA1;1, Lyces;CycA2;1, Lyces; CycA3;1, Lyces;CycB1:1 and Lyces;CycB2;1 encoding tomato mitotic cyclins, and Lyces;CycD3;1 encoding a G1 cyclin. The accumulation of transcripts was predominantly associated with mitotically active organs: developing fruits, young leaves and roots, and with cell-suspension cultures under appropriate sugar feeding conditions. Transcripts for all the isolated cyclin genes could be detected in the epidermis and pericarp of fruit tissues where some slight mitotic activity still remained at the onset of ripening. However, Lyces;CycA3;1 and Lyces;CycD3;1 were expressed in the gel tissue at the late stage of fruit development, suggesting that they are involved in endoreduplication of the differentiated and giant cells of the gel tissue.

Amino Acid Sequence↗

Regulation of cell expansion by the DISTORTED genes in Arabidopsis thaliana: actin controls the spatial organization of microtubules.

The control of the directionality of cell expansion was investigated using a class of eight genes, the so-called DISTORTED (DIS) genes, that are required for proper expansion of leaf trichomes in Arabidopsis thaliana. By tracing the separation of latex beads placed on the trichome surface, we demonstrate that trichomes grow by diffuse rather than tip growth, and that in dis mutants deviations from the normal orientation of growth can occur in all possible directions. We could not detect any differences in intracellular organization between wild-type and dis-group mutants by electron microscopy. The analysis of double mutants showed that although the expression of the dis phenotype is generally independent of branching and endoreduplication, dis mutations act synthetically in combination lesions in the ZWI gene, which encodes a kinesin motor protein. Using a MAP4:GFP marker line, we show that the organization of cortical microtubules is affected in dis-group mutants. The finding that most dis-group mutants have actin defects suggested to us that actin is involved in organizing the orientation of microtubules. By analyzing the microtubule organization in plants treated with drugs that bind to actin, we verified that actin is involved in the positioning of cortical microtubules and thereby in plant cell expansion.

Actins↗

Bovine trophoblastic cell differentiation on collagen substrata: formation of binucleate cells expressing placental lactogen.

The differentiation of trophectoderm in ruminants is marked by the appearance of binucleate cells in cytotrophoblasts. Binucleate cells are produced by the acytokinesis of cytotrophoblasts and undergo endoreduplication. They secrete hormones such as placental lactogen, and exhibit migratory behavior to transfer their hormones into maternal circulations. In this study, we showed that a bovine trophoblastic cell line (BT-1) established from in vitro fertilized blastocysts differentiated into binucleate cells on collagen gel. BT-1 had cytotrophoblastic epithelial characteristics in that it expressed cytokeratin, E-cadherin and interferon-tau. It spontaneously formed multicellular spherical vesicles floating in the medium. We cultured these vesicles on type I collagen substrata. Most vesicles attached to the collagen substrata, and exhibited cell outgrowth and proliferation. We found that after more than 10 days, clusters of binucleate cells appeared in the cell colonies on the collagen gel, but not on the collagen film. These binucleate cells have features characteristic of those in vivo, including an increased nuclear DNA content and the expression of placental lactogen. BT-1 is a useful model with which to study trophoblast differentiation in ruminants.

Animals↗