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PubMed2002

Signaling by the Mpl receptor involves IKK and NF-kappaB.

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.

Animals↗
PubMed2006

Functional characterization of hNUDC as a novel accumulator that specifically acts on in vitro megakaryocytopoiesis and in vivo platelet production.

Human NUDC (hNUDC) has been previously described as a human homolog of a fungal nuclear migration protein. It is a multifunctional interactive protein that forms an association with the microtubule motor complex in a variety of cells. Our recent studies demonstrated that hNUDC could bind specifically to the thrombopoietin receptor (Mpl) and suggest a potential role for hNUDC in megakaryocytopoiesis and thrombopoiesis. The present study is designed to define its biological activity. We demonstrate that the recombinant hNUDC significantly increases megakaryocyte maturation in serum-free liquid-cultured human CD34(+) cells and stimulates colony formation in serum-free semi-solid cultures. Flow cytometry analyses also confirm the stimulatory effect of hNUDC on megakaryocyte polyploidization and in vitro platelet production. In vivo experiments further demonstrate that the administration of hNUDC substantially enhance the number of circulating platelets in normal mice.

Animals↗
PubMed1993

Pathological and biological relevance of cytophotometric DNA content to breast carcinoma genetic progression.

Correlating cytophotometrically detectable genetic alterations to events of known biological and pathological significance in breast carcinoma has been challenging, in large part owing to the difficulty in isolating and analyzing premalignant (i.e., hyperplastic) or preinvasive (i.e., in situ carcinoma) lesions. This problem may be addressed by using histologically directed evaluation of intact, paraffin-embedded tissue sections. Using image cytophotometry in preserved sections, we have identified clonal DNA content abnormalities (i.e., aneuploidy) in up to three-fourths of preinvasive breast carcinomas. Moreover, comparison of ploidy determinations between residual in situ and corresponding invading neoplastic populations suggests that host invasion is accompanied by measurable DNA content shifts in many cases. Image cytophotometric DNA content abnormalities are also detectable in florid/atypical proliferative lesions, albeit less frequently (-25% of cases) and to a lesser extent (i.e., near-diploid) than in situ carcinomas. Taken together, these findings imply an association between clonal DNA content aberrations and histologic disease progression. Although the sensitivity of cytophotometric ploidy assessments in tissue sections is limited by nuclear sectioning artifact and overlap, the presence of genomic instability in precursor lesions is supported by evidence of individual chromosome aneuploidy, which can be demonstrated in tissue sections by interphase cytogenetics with fluorescent, centromere-specific probes. Further, presence of intra-tumoral clonal DNA content heterogeneity is confirmed by cytogenetic studies showing co-existing near-diploid chromosome number modes in many tumors with hyperdiploid stemlines. Karyotypic stemline analyses imply polyploidization events are an important mechanism of clonal evolution leading to genetic heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed2002

Roads to polyploidy: the megakaryocyte example.

Polyploidy, recognized by multiple copies of the haploid chromosome number, has been described in plants, insects, and in mammalian cells such as, the platelet precursors, the megakaryocytes. Several of these cell types reach high ploidy via a different cell cycle. Megakaryocytes undergo an endomitotic cell cycle, which consists of an S phase interrupted by a gap, during which the cells enter mitosis but skip anaphase B and cytokinesis. Here, we review the mechanisms that lead to this cell cycle and to polyploidy in megakaryocytes, while also comparing them to those described for other systems in which high ploidy is achieved. Overall, polyploidy is associated with an orchestrated change in expression of several genes, of which, some may be a result of high ploidy and hence a determinant of a new cell physiology, while others are inducers of polyploidization. Future studies will aim to further explore these two groups of genes.

Animals↗
PubMed1979

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.

Cell Cycle↗
PubMed1982

Altered cell cycle progression and aberrant mitosis in adenovirus-infected rodent cells.

Actively growing mouse or rat embryo cells suffered structural chromosome damage, mitotic anomalies, and polyploidy after infection by human adenovirus type 5. Chromosome damage required expression of one or more early viral genes and showed regular periodicity in its frequency. The growth cycle time of some of the infected cells was reduced by about 5 hours due to a decrease in G1, and the interval between successive waves of chromosome damage corresponded to this reduced cycle time. After infection there was a decrease in cells with G1 DNA content and an increase in cells with G2 diploid, aneuploid, and polyploid DNA contents. We suggest these effects are due to the expression in semipermissive cells fo early viral gene(s), whose function in productive infection in vivo is to alter cell cycle controls in order to maximize the number of cells able to replicate viral DNA and the time such cells spend in DNA replication.

Adenoviridae Infections↗
PubMed1983

Bovine endothelial cells transformed in vitro by benzo(a)pyrene.

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed lines with altered phenotypic characteristics. The transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid. Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid karyotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 10(7) cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.

Animals↗
PubMed1985

Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts.

When randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.

Animals↗
PubMed1992

Karyotypic and phenotypic changes during in vitro aging of human endothelial cells.

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture-related changes, senescence, cardiovascular disease, and tumorigenesis.

Adult↗
PubMed1994

IL-3 and ribavirin induce high level expression of megakaryocytic markers and messages during long-term treatment of a megakaryocytic leukemia cell line.

Megakaryocyte differentiation is a lengthy process with cells moving through a continuum delineated by the sequential expression of specific gene products. The limited number of primary cells available from marrow for analysis has brought attention to some leukemic cell lines which show enhanced megakaryocyte marker expression following incubation with inducing agents, the most common of which is phorbol myristate acetate (PMA). We developed an alternative induction protocol for the megakaryocytic leukemic cell line CMK, which involved incubation of the cells with IL-3 and the nucleoside analog, ribavirin, for 1-2 weeks. This treatment was neither toxic nor cytostatic and yielded increased levels of the surface glycoproteins GPIIb/IIIA and GPIb-IX. Levels of some megakaryocytic messages (GPIIIa, GPIX) showed a marked rise by 12 days of incubation in the inducer combination. This was due to a synergistic interaction between IL-3 and ribavirin which influenced both transcriptional and posttranscriptional events. Light and electron microscopy demonstrated the presence of large polyploid cells, with morphological features similar to those of megakaryocytes, in the induced cultures. Analysis of the heterogeneity of response in the cell population to the induction regimen after several days of treatment suggested that cells which failed to display surface markers had been stimulated by the inducers but did not have sufficient time to complete expression of that marker. The results were consistent with the view that the cells in the starting population were distributed along a temporal expression pathway, and those which were first to express the earliest marker would also lead in the expression of a later marker. The order of expression was the same as that during normal megakaryocyte development.

Antigens, Surface↗
PubMed1995

IL-3 and ribavirin induce differentiation and growth suppression during long-term treatment of a megakaryocytic leukemia cell line.

Differentiation of the megakaryocytic leukemia cells, CMK, was induced by long-term (12 day) treatment with the combination of IL-3 and the nucleoside analogue ribavirin (RV), which reduces cellular GTP levels. In a previous report we demonstrated the induction of early messages and antigens, as well as the formation of giant polyploid cells in the cultures (Majumdar et al., 1994, J. Cell. Physiol., 160:29-39). Here we show high level induction of messages for the late markers, Platelet Factor 4, GMP140 (P-Selectin), thrombospondin, and beta thromboglobulin. The induced cells are also positive for these antigens by immunocytochemical analysis. The high level message induction resulted from synergy between the inducers. Pretreatment of the cells with IL-3 could accelerate the rise in message seen with the inducer combination. The increase in differentiation markers was accompanied by a reduction of the proliferative capacity of the cells. Riboguanosine, which has anti differentiation activity, blocked the induction of early and late antigens by the inducer combination, and also by IL-3 acting alone, but did not block the reduction in proliferative competence. In this model of megakaryocytic differentiation IL-3 treatment yields an initial stimulation of growth followed by growth suppression, and is the principal driver of the differentiation process. RV functions primarily as a stimulator of message and protein expression in synergy with IL-3.

Cell Adhesion Molecules↗
PubMed2005

Isolation of a stromal cell line from an early passage of a mouse mammary tumor line: a model for stromal parenchymal interactions.

We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.

Animals↗
PubMed1993

Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst.

Prominent among the various types of cell that differentiate from the trophectoderm of the mouse blastocyst are trophoblastic giant cells. Repeated endoreduplication of the genome accompanies the growth of these cells, which have been shown to be polytene rather than polyploid. Early stages in giant transformation have been examined, mainly in the mural trophectoderm of the implanting blastocyst which gives rise to the primary trophoblastic giant cells. One confusing issue is whether these early stages include the onset of endoreduplication of the genome. This issue has been addressed in the present study by comparing the DNA content of nuclei in isolated trophectoderm and ICM tissue rather than, as previously, by relating measurements on air-dry preparations of entire blastocysts to those of adult liver. The results, particularly those from delayed and reactivated blastocysts, show that genome endoreduplication is not an obligatory early event in the transformation of mural trophectoderm cells.

Animals↗
PubMed2004

Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae).

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.

Animals↗
PubMed2004

Simultaneous production of triploid and haploid eggs by triploid Squalius alburnoides (Teleostei: Cyprinidae).

The hybrid minnow Squalius alburnoides comprises diploid and polyploid forms with altered modes of reproduction. In the present paper, we report a cross where a triploid female generated both large, triploid and small, haploid eggs simultaneously, which were fertilized with S. pyrenaicus sperm. Although the large eggs were rarer (15%), they originated offspring with higher survivorship, so that tetraploids were dominant among the surviving siblings. The cross yielded apparently all female progeny. Inheritance patterns were inferred using four microsatellite markers and NORs (Nucleolus Organizer Regions) phenotypes, and suggested that haploid eggs were probably produced by an atypical hybridogenesis, in which the elimination of the unmatched genome permitted random segregation and recombination between the homospecific genomes, while the triploid eggs were clonal. The present results suggest that the occurrence of triploid unreduced eggs may be a new route for the natural tetraploidization in the complex.

Animals↗
PubMed2003

Telomerase activity is widespread in adult somatic tissues of Xenopus.

Chromosome ends, or telomeres, are maintained by telomerase. Work in selected vertebrates has implied that telomerase is often repressed in differentiated cells, and telomere erosion results in senescence of cultured cells. Tissues from mature Xenopus laevis frogs were examined for telomerase enzymatic activity with the TRAP (telomere repeat amplification protocol) assay. All tissues contained active telomerase, most abundantly in testis, spleen, liver, and embryos; activity was less abundant but still readily detectable with < 100 ng of protein extract from brain and muscle tissues. Activity in somatic tissues of the diploid Xenopus tropicalis suggests this condition is not limited to the polyploid members of the genus, and that extensive differentiation-linked telomerase repression does not occur in Xenopus.

Aging↗
PubMed2005

Genome size and endonuclear DNA replication in spiders.

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

Animals↗
PubMed1989

Obplacental giant cells of the domestic rabbit: development, morphology, and intermediate filament composition.

Obplacental giant cells are large (less than or equal to 210 microns) polyploid cells that appear in the stroma of the pregnant uterus of the rabbit following ovoimplantation. Histological examination of a complete developmental series indicates that obplacental giant cells arise from trophoblastic knobs that have traversed the uterine epithelium during early implantation. During maturation, the cells undergo a massive (approximately 6,000%) increase in volume and penetrate deeply into the uterine stroma and myometrium, where they often become associated with blood vessels and smooth muscle cells. Giant cells at mid-gestation contain one or two large nuclei with prominent nucleoli and appear to be amitotic. They are rich in Golgi complexes, RER, SER, and cortically distributed cytoplasmic filaments, and contain intracellular canaliculi lined by microvilli. Giant cells vary with respect to the occurrence of lipid droplets, phagocytotic inclusions, lysosomal structures, and electron-dense granules. Immunocytochemistry demonstrates that the giant cells exhibit intermediate filaments related to cytokeratin and vimentin, but are negative for desmin and for an endothelial cell marker, Factor VIII-related antigen. The cells are positive for cytokeratin from their inception, but only become vimentin-positive between Days 12 and 15 of pregnancy, a change seemingly related to their detachment from epithelial tissue to take on an independent existence. Our findings indicate that the giant cells originate from obplacental trophoblast and, at maturity, exhibit cytoskeletal characteristics of isolated epithelial cells, as well as a complement of organelles suggestive of synthetic activity.

Animals↗