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Whole-body X-irradiation of mice accelerates polyploidization of hepatocytes.

Male C57BL/6 mice were whole-body irradiated with 4.75 Gy of X-rays at the age of 2 months and killed at 2, 6, 12 and 19 months after irradiation. The percentage survival began to decline earlier and faster in the irradiated group than the controls up to 19 months after exposure when the study was terminated. The nuclear DNA content of individual hepatocytes was measured by a Feulgen-DNA microfluorometric method, and hepatocytes were classified into various ploidy classes. In the irradiated mice, the degree of polyploidization was significantly higher than the controls by 2 months after exposure and steadily increased up to 6 months after exposure. Thereafter, however, a slow return to the control level was found up to 19 months after irradiation. These results appear to support a hypothesis that radiation accelerates the ageing process as judged from hepatocyte polyploidization.

Aging

The level of aryl hydrocarbon (Ah) receptor and of 4S polycyclic aromatic hydrocarbon (PAH) binding protein in diploid and polyploid hepatocytes of 2-acetylaminofluorene-treated rats.

Sequential treatment of partially hepatectomized male Wistar rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) induces the emergence of diploid hepatocyte populations. These carcinogen-induced hepatocytes are thought to include the precursor cells of liver carcinomas that arise later in this treatment protocol. The growth of the diploid hepatocytes is promoted by AAF and it has been suggested that the action of the arylamine may be receptor-mediated. AAF has been shown to bind specifically to the aryl hydrocarbon (Ah) receptor and the so-called 4S polycyclic aromatic hydrocarbon (PAH) binding protein. The present study addresses the question of whether the concentrations of the two binding proteins differ in diploid and polyploid hepatocytes from DEN/AAF-treated rats. Hepatocytes from carcinogen-treated rats were isolated and diploid, and tetraploid hepatocytes separated by means of centrifugal elutriation. Whereas Ah receptor concentrations in diploid hepatocytes were insignificantly lower (21.8 +/- 5.9 versus 29.2 +/- 6.6 fmol/mg cytosolic protein; n = 4; P = 0.1), levels of the 4S PAH binding protein in diploid hepatocytes were twice as high as in tetraploid hepatocytes (252.3 +/- 93.6 versus 124.0 +/- 18.5 fmol/mg cytosolic protein; n = 4; P = 0.04). We conclude from our results that the differences in growth control in polyploid and carcinogen-induced diploid hepatocytes are not associated with changes in the levels of the Ah receptor. The role of the 4S PAH binding protein in the process of hepatocarcinogenesis remains to be established.

2-Acetylaminofluorene

The polyploidizing growth pattern of normal rat liver is replaced by divisional, diploid growth in hepatocellular nodules and carcinomas.

DNA content was measured by flow cytometry in isolated nuclei from 71 neoplastic nodules and 15 hepatocellular carcinomas isolated from rat liver at various times after treatment with an initiation--promotion regimen employing diethylnitrosamine and 2-acetylaminofluorene. Nodules and carcinomas contained mostly diploid nuclei as compared with both surrounding and normal hepatocytes which were predominantly polyploid. There appears to be a positive correlation between the degree of diploidy in nodules and their rate of proliferation. No aneuploid populations were identified in any neoplasm despite good peak resolution. These results show that an alteration in proliferation pattern from normal polyploidizing growth to diploid--diploid divisional growth is a consistent characteristic throughout the carcinogenic process in our experimental model.

2-Acetylaminofluorene

Diploids derived from polyploids: genetic characteristics of four novel interspecific Sorghum populations.

Polyploidy has repeatedly shaped grass evolution, yet direct observations of how polyploid-derived chromosomes behave when returned to diploidy remain rare. Interspecific crosses between diploid Sorghum bicolor and tetraploid hybrids derived from Sorghum halepense generate mixed-ploidy progeny, providing an opportunity to examine chromosome transmission during the early stages of diploidization. Using genome-wide SNP markers, we characterized chromosomal inheritance patterns in 2 diploid and 2 tetraploid families derived from these crosses. Genotype-dosage profiles alone distinguished diploids from tetraploids with complete accuracy, reflecting strong ploidy-dependent differences in dosage-class distributions. Although diploid progeny retained much of the halepense-derived genomic background, several genomic intervals exhibited extended, nonrandom runs of S. bicolor homozygosity that remained polymorphic in corresponding tetraploid populations. These patterns, together with recurrent segregation distortion across independent families, suggest that the transition from tetraploidy to diploidy can expose allelic combinations that differ in transmission or viability. Analyses of flowering time further indicated that diploid and tetraploid derivatives possess distinct genomic architectures, with major association peaks occurring in different chromosomal regions across ploidy levels. Collectively, these results indicate that early diploidization involves nonrandom retention and loss of parental haplotypes shaped by both selective and structural constraints. The diploid extractions characterized here provide a rare empirical system for investigating the early stages of diploidization and a practical framework for studying and eventually mobilizing polyploid-derived variation for sorghum germplasm development. However, broader integration into elite breeding programs will require additional evaluation of cross-fertility, meiotic behavior, and chromosomal stability across diverse breeding backgrounds.

Sorghum

Excess glucose intake induces accelerated beta-cell polyploidization in normal mice: a possible deleterious effect.

Normal nondiabetic homozygous and heterozygous littermates of db/db diabetes-prone mice (seven/group) were fed a 5% solution of glucose as their sole source of liquid for 10 wk. Controls (seven/group) drank tap water, and both groups received stock diet ad libitum. Body weights, tail lengths, food and fluid consumption were recorded throughout the study, and plasma and urine glucose were measured during wk 10. The total caloric intake, including the glucose solution drunk by some of the mice, was not significantly different among the four groups. No differences in plasma or urine glucose were detected. Total-body dry weight, water and lipid were measured, and pancreata were analyzed for beta-cell polyploidy by a combination of Feulgen cytophotometry and nuclear size analysis. The percentage of polyploid beta-cells was significantly higher in the animals that drank glucose than in those that drank water and was independent of both genotype and growth indices attributable to genotype. The greater polyploidization was interpreted as reflecting premature aging of the beta-cell population. It was hypothesized that such glucose-induced premature aging in animals with a genetically restricted potential for beta-cell proliferation could contribute to the precipitation of overt diabetes.

Animals

The genome of RNA tumor viruses: a functional requirement for a polyploid structure?

This paper points out certain theoretical problems in DNA synthesis associated with antiprimer transcription and with circularization that could oblige RNA tumor viruses to rely on a polyploid genome. It is suggested that each completed act of reverse transcription may be coupled with an act of genetic recombination aimed at recovering the antiprimer information from an adjacent genome subunit in a polyploid train. A partially double-stranded DNA transcript could then be formed with sufficient terminal redundancy to permit circularization. The model provides satisfactory explanations for observed genetic interactions (particularly recombination and heterozygote formation), for inactivation data and for selective subunit transcription.

DNA, Viral

Morphological and genetic effects of benomyl on polyploid brewing yeasts: isolation of auxotrophic mutants.

An enrichment procedure after ethyl methanesulfonate mutagenesis and exposure to the fungicide benomyl yielded mutants auxotrophic for several amino acids from two polyploid Saccharomyces spp. Benomyl treatment was found to have a marked morphological effect on polyploid Saccharomyces cerevisiae, causing cells to adopt a characteristic doublet cell morphology in which buds are nearly as large as the parent cells. Experiments in which nuclear division was monitored in benomyl-induced doublet cells by Giemsa nuclear staining demonstrated an unusual sequence of cytological events which culminated in the formation of binucleate parental and mononucleate bud components. The frequency of formation of doublet and binucleate parent cells was found to depend on the strain employed and the benomyl concentration administered.

Benomyl

Biochemical data bearing on the relationship between the genome of Triticum urartu and the A and B genomes of the polyploid wheats.

To determine whether the Triticum urartu genome is more closely related to the A or B genome of the polyploid wheats, the amino acid sequence of its purothionin was compared to the amino acid sequences of the purothionins in Triticum monococcum, Triticum turgidum, and Triticum aestivum. The residue sequence of the purothionin from T. urartu differs by five and six amino acid substitutions respectively from the alpha 1 and alpha 2 forms coded for by genes in the B and D genomes, and is identical to the beta form specified by a gene in the A genome. Therefore, the T. urartu purothionin is either coded by a gene in the A genome or a chromosome set highly homologous to it. The results demonstrate that at least a portion of the T. urartu and T. monococcum genomes is homologous and probably identical. A variety of other studies have also shown that T. urartu is very closely related to T. monococcum and, in all likelihood, also possesses the A genome. Therefore, it could be argued that either T. urartu and T. monococcum are the same species or that T. urartu rather than T. monococcum is the source of the A genome in T. turgidum and T. aestivum. Except for Johnson's results, our data and that of others suggest a revised origin of polyploid wheats. Specifically, the list of six putative B genome donor species is reduced to five, all members of the Sitopsis section of the genus Aegilops.

Amino Acid Sequence

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.

Aging

Megakaryocyte polyploidization in May-Hegglin anomaly.

Polyploidization of megakaryocytes was studied in bone marrow aspirates from 3 patients with May-Hegglin anomaly by combined application of cytophotometric determination of the DNA content and autoradiography with 3H-TdR labeling in vitro. A marked elevation of the influx of progenitor cells into the megakaryocytic cell system as well as a decreased maturation capacity from type II to type III megakaryocytes was observed possibly contributing to the pathological platelet sequestration. The polyploidization activity as assessed by 3H-TdR labeling and nuclear DNA content was normal.

Adolescent

Effects of butyrate on cell cycle progression and polyploidization of various types of mammalian cells.

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Y1, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.

Animals

Aneuploid and polyploid cellular DNA heterogeneity in insect cell material of diptera species analyzed by flow cytometry.

Flow cytometric 1-parameter DNA analysis and 2-parameter DNA/protein analysis have been performed with cell material of the diptera species Chironomus thummi, Drosophila melanogaster, Calliphora vicina and Musca domestica using an impulse cytophotometer with a new quartz objective, that was especially manufactured for cytofluorometric investigations. The occurrence of heterogenous cell populations with aneuploid and polyploid DNA content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. Partially separation of 2 C cells from other cell populations with higher DNA content can be done by Ficoll-Hypaque centrifugation as demonstrated with cell material from Chironomus larvae. For flow cytometric DNA analysis of insect cell material a simple and rapid cell preparation and staining technique is presented by using the DNA-specific fluorochrome DAPI in combination with the protein fluorochrome sulforhodamine 101. Employment of flow cytometry in diptera genetics might be a new tool for cytological and cytogenetic investigations as shown with the classical genetic objects Chironomus and Drosophila.

Aneuploidy

Polyploid cells in blastocysts and early fetuses from Australian Merino sheep.

Cytogenetic examination was made of 103 13-14-day-old blastocysts and 116 24-32-day-old fetuses from untreated and androstenedione-7-HSA-immunized Merino ewes. There were no differences in the chromosome composition of blastocysts or fetuses from treated or untreated ewes and so the data were combined. At Days 13-14 a 1N/2N mosaic and a 2N - 1/2N/4N mosaic embryo were observed. In addition, 52 of the blastocysts were 2N/4N mosaics, with 8 of these also containing 8N cells, and one blastocyst was a 2N/8N mosaic. No aneuploid fetuses were observed, but 80 of the 116 fetuses contained polyploid cells, including 4N, 6N and 8N cells. The polyploid cells observed in the blastocysts and fetuses should not be considered as abnormal cells as they appear to be a normal part of the developmental processes leading to trophoblast formation and fetal differentiation.

Animals

[The induction of hepatocyte polyploidization in rats of various ages by ionizing irradiation with different LETs].

A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of nonproliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation.

Aging

[Polyploidization of rat hepatocytes due to cell fusion caused by radiations of different LET].

The method of flow cytometry was used to study polyploidization of hepatocytes following X-, gamma-, and neutron-irradiation. Ionizing radiation was shown to induce cell polyploidization by two different ways: (1) cells and nuclei fusion, and (2) restriction of mitosis after DNA replication. RBE of 14 MeV neutrons with respect to fusion was about 5.10(3). With neutron irradiation, the sensitivity of cells by fusion was not lower than that by chromosome mutations.

Animals

[Polyploidization of cardiac myocytes as a programmed event of ontogenesis].

The pieces of ventricles of newborn rats, which consist of diploid myocytes in more than 95%, were transplanted under the renal capsule of a syngeneic adult rat. Within 35 days the transplants contained 40 to 60% of the cells with the double or even greater DNA content in the mononuclear and binuclear classes. The composition of the classes was similar unlike the number of the cells of a certain class in the transplant and in the heart of a 35 day old rat. A conclusion has been drawn that the polyploidization is programmed and the realization of this programme depends on the growth, work, and other factors but is expressed even at their minimal action in the transplant. The growth of the transplanted myocytes was weaker than in the heart. It is supposed that, unlike the polyploidization, the growth of the myocytes outside the cycle is, predominantly, functional-dependent.

Animals

[Analysis of the polyploidization kinetics of the parenchymal cells in the rat liver].

Methodological approaches to kinetics of cell polyploidization in the rat liver parenchyma are discussed. Different ways of hepatocyte polyploidization in the course of postnatal liver growth have been assessed. The intensities of hepatocyte transitions from one ploidy class to another were determined. On the basis of literary experimental data the following is summarized: With the increase in the animal age, there is a decrease in hepatocyte transition from one ploidy class to and ther; in young animals the intensity of formation of tetraploid hepatocytes through the stage of binuclear cells (2c----2c X 2----4c) is 0.39-0.55 within two weeks, the intensity of direct transitions (2c----4c) being 0.00-0.19 within the same time. The intensity of entering to DNA synthesis is reduced with the increase in hepatocyte ploidy levels; in this case the coefficient of the reducing of mitotic activity is calculated as 0.10-0.22, and 0.01-0.05 for 4c- and 8c-hepatocytes, resp. The factors stimulating proliferation in the liver increase the intensity of the direct cell transition (2c----4c) by several times which can exceed the intensity of transition through the binuclear cell stage.

Aging