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PubMed2005

[Evidence of the unreduced diploid eggs generated from the diploid gynogenetic progeny of allotetraploid hybrids].

Following activation by UV-irradiated sperms from scatter scale common carp and without the treatment for doubling the chromosome number, the eggs generated by the diploid gynogenetic progeny (G1) with 100 chromosomes,developed into diploid gynogenetic progeny (G2) with 100 chromosomes. Both the males and females of the tetraploids with 200 chromosomes were obtained from the offsprings (G1 x AT) produced by mating the eggs of G1 with the diploid sperms from the allotetraploid hybrids. The results provided the evidence that diploid G1 is able to produce diploid eggs. In the oogonia of diploid six-month old G2, only the chromosomes in the metaphase of mitosis were observed in all slides, no bivalent chromosome being found, suggesting that the oogonia in six-month old G2 were not mature enough for meiosis I. The presence of the chromosome spreads with 100, 200 and 380 chromosomes observed in the metaphase indicated that the diploid G2 had the potential ability to generate diploid eggs with the probable mechanism of pre-meiotic endoreduplication. In contrast, the spermatocytes of G1 x AT allotetraploids had the normal chromosomal behavior with only 100 bivalents found in meiosis I. The one-year old G2 possessed the slowly developmental ovaries which stayed at the oogonium stage for a long time, in which no primary and mature oocyte was found, whereas both the females and males of one-year old G1 x AT allotetraploids had normal ovaries and testes which reached maturity and produced diploid eggs and diploid sperms, respectively. The formation of the diploid eggs generated from the diploid gynogenetic progeny makes it available to establish a diploid hybrid clonal line, and will become an important source of production of diploid eggs. In addition, the bisexual fertile G1 x AT allotetraploids enriched the types of the tetraploids.

Animals↗
PubMed2005

Genotoxic effects of rotenone on cultured lymphocytes.

Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle.

Adult↗
PubMed1990

[Proliferation and differentiation in megakaryopoiesis].

Megakaryocytic progenitor cells only complete a limited number of mitotic events; early in megakaryopoiesis, these cells loose their capacity for cell division and acquire the ability for endoreduplication--a phenomenon that is unique to the megakaryocytic lineage. There is growing evidence, that at least two humoral activities affect proliferation of progenitor cells and maturation of its progeny. However, the cytokines that mediate these functionally defined, overlapping activities are not yet precisely known. This review summarizes current understanding of these processes and will discuss the interactions of megakaryocytic cells with the bone marrow microenvironment as well as their role in disorders of hematopoiesis.

Animals↗
PubMed1990

Heterochromatization and euchromatization of whole genomes in scale insects (Coccoidea: Homoptera).

In several families of scale insects (coccids), the sex of an embryo is determined by the number of genetically active genomes present (one = males, two = females). In mealybugs (Pseudococcidae), both males and females develop from fertilized eggs but, in the embryos that develop into males, the set of chromosomes (genome) of paternal origin (PG) becomes heterochromatic (H) and genetically inactive and is not transmitted to the offspring. The mechanism that reduces the number of active genomes in male embryos may vary between families and even between congeneric species. Thus, in male embryos of most armored scale species (Diaspididae), the PG is eliminated, while in a few species it becomes H. In two genera of soft scales (Coccidae), males develop from unfertilized eggs when one of two identical genomes of maternal origin becomes H. In most male tissues, one genome remains H. However, in several tissues that become polyploid by endoreduplication, the PG becomes E and genetically active. The tissues in which the PG becomes E often vary between species and the analysis of hybrid males demonstrated that whether the PG becomes H or remains E is determined by the genome of maternal origin. The euchromatization of the PG in the haploid sector of mosaic male embryos and the presence of spermatocytes with two E genomes (instead of one E and one H), following the irradiation of young mealybug males, strongly suggest that the maintenance of the H state requires the presence of a genetically active genome.

Animals↗
PubMed1990

Mitotic disturbances associated with inversion 9qh. A case report.

A 25-year-old female with history of spontaneous abortion and subsequent birth of Down syndrome child was referred for chromosome analysis. Her karyotype revealed 46, XX with pericentric inversion of 9 qh, while her husband was normal with 46, XY chromosomes. Metaphase analysis of the female showed 20.5% cells with premature centromere division, 4% with endoreduplication, 2% with polyploidy and 9.33% aneuploidy. These frequencies were considerably higher as compared to a normal control. These observations suggest that inv (9qh) might have some interchromosomal effect leading to higher incidence of mitotic disturbances, finally resulting in aneuploidy. This predisposition is evident by spontaneous abortion and later birth of a Down syndrome child.

Abortion, Habitual↗
PubMed1990

DNA and sex chromatin content in nuclei of conductive system and working myocytes of normal and hypertrophied human heart.

DNA content was measured by means of Feulgen cytophotometry in myocyte nuclei of sinoatrial und atrioventricular nodes, Purkinje cells, left and right atria and ventricles from normal and hypertrophied human hearts both on 12 microns sections and on squash preparations of myocardial tissue. In contrast to ordinary atrial and ventricular myocytes of normal and hypertrophied adult hearts, which contain mostly polyploid nuclei, the DNA content in 74 to 88.5% of myocyte nuclei from sinoatrial and atrioventricular nodes does not exceed 2 c. Unlike nodal myocytes in Purkinje cells of hypertrophied adult heart there are 97 to 99.5% of polyploid nuclei, 49 to 88% of them displaying octaploid and higher DNA values. Myocytes of highly hypertrophied atria contain nuclei of considerably higher ploidy level than those of normal atria. Percentage of binucleated cells show a 4-fold increase in hypertrophied atria compared with normal ones. The total area of nucleoli per nucleus is proportional to the degree of ploidy. Number of sex chromatin bodies counted simultaneously with DNA measurements on sections of atrioventricular node and on squash preparations of atria and ventricles is correlated with ploidy level. Judging by correlations between the number of sex chromatin bodies and DNA content in nuclei, on one hand, and between the total area of nucleoli per nucleus and the degree of ploidy, on the other, the mitotic endoreduplication (true polyploidization) is responsible for the increased DNA content in nuclei of conductive system, atrium and ventricle.

Adult↗
PubMed1990

Genetics and biology of human ovarian teratomas. I. Cytogenetic analysis and mechanism of origin.

One hundred and two benign, mature ovarian teratomas and two immature, malignant teratomas were karyotyped and scored for centromeric heteromorphisms as part of an ongoing project to determine the chromosomal karyotype and the genetic origin of ovarian teratomas and to assess their utility for gene-centromere mapping. Karyotypic analysis of the benign cases revealed 95 46,XX teratomas and 7 chromosomally abnormal teratomas (47,XXX, 47,XX,+8 [two cases], 47,XX,+15, 48,XX,+7,+12 91,XXXX,-13 [mosaic], 47,XX,-15,+21,+mar). Our study reports on the first cases of tetraploidy and structural rearrangement in benign ovarian teratomas. The two immature cases had modal chromosome numbers of 78 and 49. Centromeric heteromorphisms that were heterozygous in the host were homozygous in 65.2% (n = 58) of the benign teratomas and heterozygous in the remaining 34.8% (n = 31). Chromosome 13 heteromorphisms were the most informative, with 72.7% heterozygosity in hosts. The cytogenetic data indicate that 65% of teratomas are derived from a single germ cell after meiosis I and failure of meiosis II (type II) or endoreduplication of a mature ovum (type III); 35% arise by failure of meiosis I (type I) or mitotic division of premeiotic germ cells (type IV).

Adolescent↗
PubMed1979

In vivo and in vitro studies on the early embryonic lethal oligosyndactylism (Os) in the mouse.

The development of mouse embryos homozygous for oligosyndactylism (Os) is arrested during implantation. Histological investigations confirm a previous report that cells become blocked in mitosis, and air-dried spreads of the mutant embryos reveal that large numbers of cells accumulate in metaphase. Trophoblastic giant cells appear unaffected by the action of the mutant gene both in utero and during culture over the lethal phase. It is proposed that the form of endoreduplication undergone by giant cells renders them refractory to the metaphase block.

Animals↗
PubMed1990

Effects of 1-beta-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution.

The pH-step alkaline elution method enables the isolation and quantification of nascent DNA (nDNA) replication intermediates, including Okazaki fragments, short length nDNA from replicon origins, longer lengths of nascent but subgenomic length nDNA (molecular weight, 20-30 x 10(6)), and full (or genomic) length nDNA (L. C. Erickson et al., Chromosoma, 74: 125-139, 1979). We utilized this technique to study, in HL-60 cells, the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on the formation of these replication intermediates and the kinetics of transit of radiolabel from [3H]thymidine ([3H]dThd) or [3H]-ara-C through these nDNA fragments and into full length nDNA. In the continuous presence of [3H]-ara-C (4 microM), the majority of radiolabel (greater than 85%) remained in the nascent subgenomic fractions, with 30-50% remaining in Okazaki fragments. These proportions did not change substantially with increasing time of exposure to [3H]-ara-C (from 1 to 24 h), although the total amount of [3H]-ara-C incorporated into DNA continued to increase with increasing time of exposure. In contrast, when cells were exposed to [3H]-ara-C for 1 h, placed in drug-free medium, and studied by the pH-step method at various times thereafter, the transit of radiolabel through progressively larger nDNA intermediates and into full length nDNA was rapid and equal to that of [3H]dThd in cells not exposed to drug. The observed elution of [3H]-ara-C in the subgenomic-length DNA fragments was not due to ara-C-induced breaks in template (parental) DNA and subsequent incorporation of [3H]-ara-C into the template strand, since ara-C treatment of cells prelabeled with [14C]dThd failed to cause substantial elution of the 14C label at the various pH steps used. In studies of the effects of ara-C on [3H]dThd incorporation into nDNA, concentrations of 1 to 10 microM ara-C inhibited total incorporation of radiolabel into DNA by greater than 90% and incorporation into full length nDNA by greater than 97%. In contrast, these concentrations of ara-C failed to decrease the amount of [3H]dThd incorporated into Okazaki fragments or other non-mitochondrial low molecular weight nDNA, compared to control. These studies demonstrate that, in HL-60 cells, ara-C causes profound inhibition of nascent chain elongation, does not cause chain termination, and does not inhibit initiation. In fact, ara-C may stimulate initiation, leading credence to recent theories proposing endoreduplication or reinitiation as consequences of ara-C incorporation into DNA.

Arabinonucleotides↗
PubMed1990

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry.

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

Animals↗
PubMed1989

Is isochromosome i(12p) present in gonadal precancerous tissue?

The development of frequent gonadal tumors in some syndromes of abnormal sexual differentiation is often preceded by carcinoma in situ. To detect possible early changes in chromosome number and/or structure associated with the carcinoma in situ, gonadal tissue was studied in one patient with the complete form of testicular feminization syndrome, two patients with the incomplete form of this syndrome, and one patient with Swyer's syndrome. In all patients aneuploidy with a bimodal distribution of chromosome numbers with the peaks at diploid and near-tetraploid values was shown. The possible specific chromosomal marker of gonadal tumors, isochromosome i(12p), has not been found. Its absence suggests that this marker might be associated with more advanced stages of long-term cancer development or it could be confined only to a subgroup of testicular tumors, as was proposed earlier. The hypothesis that the major source of hypotetraploid or near-triploid cells often found in many gonadal tumors might be endoreduplications with their subsequent chromosome loss is supported.

Adolescent↗
PubMed1989

Karyotypic evolution of a human undifferentiated large cell carcinoma of the lung in tissue culture.

Serial cytogenetic studies were performed on a cell line derived from a pleural effusion from a patient with undifferentiated large cell carcinoma of the lung. The initial sample had a broad range of chromosome numbers per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid sideline. A sequence consisting of a doubling of chromosome number per cell followed by chromosome loss was observed repeatedly during 40 culture passages. The presence of metaphase spreads showing evidence of endoreduplication suggested this as a likely mechanism for the doubling of chromosome number per cell. Eleven marker chromosomes were observed in the cells of the primary sample; these markers persisted through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16 were consistently overrepresented; each of these chromosomes was involved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22 were consistently underrepresented. Every chromosome, either in its normal form and/or as part of a marker, was represented on the average by at least one copy per diploid cell. Eighteen new marker chromosomes were observed during the course of cell cultivation; one of these evolved into a clonal marker over the course of six cell passages. Of the new marker chromosomes that were formed during the observation period, the majority were found in hypotetraploid cells.

Carcinoma↗
PubMed1988

[Presence in human lymphocytes of a protein specifically binding to a cloned fragment of human satellite DNA III].

PHA-stimulated human lymphocytes contain the protein (SBP) which has selectivity in binding of 1.8 kb fragment of human satellite DNA III (HS3) as compared to other DNA sequences. It is shown that the binding site is localized within 1kb Sau3A-EcoR I fragment of HS3. SBP-binding activity is increased after treatment of cells with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The essential increase in a number of metaphases with chromosome endoreduplications in TPA-treated lymphocytes indicates that SBP may be involved in initiation of chromosome replication or in alteration of the mitotic spindle function.

Cloning, Molecular↗
PubMed1986

Cytogenetic effects of etoposide (VP-16) on human lymphocytes; with special reference to the relation between sister chromatid exchange and chromatid breakage.

The effects of etoposide (VP-16) on sister chromatid exchange (SCE) and chromosome abnormalities were studied by using human peripheral lymphocytes. This drug produced a significant increase in the SCE frequency and chromosomal aberrations such as breaks, exchanges, and tetraploidy with or without endoreduplication. Analysis of the breakpoints of chromatid deletion with respect to their association with SCE in the metaphases from the second division demonstrated that VP-16-induced deletions arose with little dependence on the site of SCE. It is suggested that chromatid deletions induced by this drug derive from unrepaired chromatid breaks rather than from incomplete exchanges.

Cells, Cultured↗
PubMed1988

Growth characteristics of circulating hematopoietic progenitor cells from patients with essential thrombocythemia.

Peripheral blood mononuclear cells from five patients with essential thrombocythemia (ET) were cultured in vitro to evaluate restricted megakaryocytic (CFU-Meg), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell development. Varying concentrations of aplastic canine serum served as the source of megakaryocyte colony-stimulating activity, and cultured megakaryocyte ploidy distributions were determined by Feulgen staining and microfluorometry. Megakaryocyte colony growth was strikingly abnormal in all five patients evaluated. Four of the 5 had a marked expansion in the concentration of circulating CFU-Meg and 3 of 4 manifested abnormalities in cultured megakaryocyte colony size (2 unusually large and 1 small). Unstimulated megakaryocyte colony growth was substantially increased in three patients. However, the fraction of megakaryocyte progenitors in cell cycle was near or below normal in all instances. Endomitotic megakaryocyte development was disordered in each of the four ET patients in whom it was evaluable. In normal subjects, mean megakaryocyte ploidy values vary biphasically with aplastic canine serum concentration and peak at 13.2 N following 12 to 15 days of culture. In contrast, day 12 mean ploidy values in cultures from the ET patients remained low at all aplastic canine serum concentrations and reached a maximum averaging only 8.4 N. Three patients were evaluated serially at extended culture durations of up to 21 days. The cultured megakaryocyte ploidy was unchanged during this interval for two of the patients. For the third patient, ploidy increased steadily, reaching abnormally high ploidy values by day 21. Progenitor cell expansion was limited to the megakaryocyte line in three patients. However, two patients had substantial increases in CFU-GM, one of whom also had a marked increase in BFU-E. There was no significant unstimulated colony growth by either CFU-GM or BFU-E. These data indicate that ET is usually characterized by an expansion in the concentration of circulating CFU-Meg in vivo which manifest both disordered replication and endoreduplication in vitro.

Cell Division↗
PubMed1987

Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells.

Recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) has been previously demonstrated to stimulate colony formation from human myeloid, erythroid, and multipotential stem cells. In this investigation, we evaluated the effects of rGM-CSF on colony growth by human megakaryocyte progenitors (CFU-Meg). rGM-CSF was tested at concentrations of 0.1-100 U/ml in plasma clot cultures of adherent-depleted normal peripheral blood mononuclear cells. Control cultures were concurrently prepared containing either no stimulator or megakaryocyte colony-stimulating factor (Meg-CSF) partially purified from aplastic canine serum. rGM-CSF increased megakaryocyte colony numbers from a baseline of 4.3 +/- 1.4 (+/- SEM) in the unstimulated cultures to a maximum of 21.0 +/- 5.3 colonies at an rGM-CSF concentration of 1.0 U/ml. Corresponding megakaryocytic colony size increased from 4.4 to 8.3 cells/colony. Further increasing the rGM-CSF concentration resulted in decreasing megakaryocyte colony growth, reaching 6.8 +/- 2.9 colonies at 100 U/ml. The maximum number of megakaryocyte colonies stimulated by rGM-CSF was only 23.3% of that achieved in the control cultures containing optimal concentrations of serum-derived Meg-CSF protein (2.0 mg/ml). Megakaryocyte colonies stimulated by rGM-CSF consisted of predominantly low ploidy cells approximately equally distributed in 2N, 4N, and 8N ploidy classes. There was no increase in ploidy with any rGM-CSF concentration. These data indicate that rGM-CSF has modest activity in stimulating human megakaryocyte colony growth that is substantially less than that present in serum-derived Meg-CSF. rGM-CSF appears to primarily affect the early mitotic phase of megakaryocyte colony development with little influence on megakaryocyte endoreduplication.

Cell Division↗
PubMed1987

Interrelationship between mitosis and endomitosis in cultures of human megakaryocyte progenitor cells.

Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5-fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.

Adult↗
PubMed1987

Growth and metastasis of hamster melanoma following transplantation into athymic mice.

Melanotic hamster malignant melanoma (MM1) is a transplantable, locally invasive tumor which metastasizes widely in syngeneic hosts. We have established three clones, HM1, HM3, and HM4 of the original MM1 line in culture. Inoculation (s.c.) into 4- to 5-week-old male athymic mice produced highly vascular, melanotic, locally invasive tumors in 100% of mice inoculated with a latency of 4-7 days. Karyotype analysis of HM cells revealed modal chromosome numbers of 39-41 (43% HM1), 43 (22% HM3), and 44-47 (61% HM4). Sixty-one % of HM1 cells were hypodiploid, 4% diploid, and 5% hyperdiploid. HM1, -3, and -4 cells also exhibited aneuploidy, endoreduplication, translocational exchanges, additions, deletions, dicentromeric and ring chromosomes, and double minutes although not all cells exhibited all abnormalities. Initial metastasis was to regional lymph nodes with eventual progression to lung and liver. Mice inoculated with HM1, -3, and -4 cells were dead with metastatic disease within 57, 63, and 64 days, respectively, following s.c. inoculation (5 X 10(5) cells) when the mice were 90-100 days old. Mortality rate was highest in line HM3 with 50% of the mice dead within 33 days postinoculation. Metastatic potential of HM1 and HM3 cells rose significantly when successive generations of HM1 and HM3 cells cultured from isolated lung metastasis were reinoculated. Metastasis to lymph nodes and liver was not observed with increasing passage generations of lung metastasis. Our observations provide evidence that hamster melanomas are clonally heterogenous, locally invasive, and exhibit rapid growth and metastasis following s.c. inoculation into adult athymic mice. Transplantable melanotic hamster melanoma cells also exhibit a significant preferential metastasis to lung following culture and sequential reinoculation of lung metastasis in athymic mice. As such, they appear to provide a reproducible model of metastasis in an immunocompromised host.

Animals↗