Origin and evolution of binucleate cells in cultures of HEp-2 cells

Abstract. The origin and evolution of binucleate cells in cultures of HEp-2 cells have been studied by means of interval photography and time-lapse video-recording. Binuc leate cells most frequently formed by the fusion of two sister cells born in a previous mitosis. The study of binucleate cells has shown that they are a cellular type able to successfully undergo mitosis. However, the mitosis may be bipolar, tripolar or multi-polar. The daughter cells arising from these divisions do not follow a clear pattern in the number of nuclei they have, instead showing a wide range of possibilities.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

Cell cycle duration and time of DNA synthesis in binucleate cells induced inV. faba meristems by caffeine or isobutyl-methylxanthine

Summary Lateral roots ofVicia faba were treated with a solution of 5-aminouracil (3.93×10^–3M) for 6 hours. After 15 hours roots were recovering from the temporary inhibition of mitosis induced by 5-AU and were approaching peak mitotic indices; they were then treated with 0.1% caffeine or 0.1% isobutylmethylxanthine (IBMX) for 1 hour. Treatment with methylxanthines when the mitotic index was high gave relatively high yields of binucleate cells, 3.8 to 7.5%. DNA synthesis, cell cycle duration and nuclear growth were determined for binucleate cells. Caffeine induced binucleate cells underwent a marked reduction in nuclear volume, from 1,074 m³ at 1+1 hours to 534 m³ at 1+14 hours. Only 15% of these binucleates entered S phase; those that did so were in mitosis or had divided by 1+14 hours. We conclude that 85% of the binucleate cells are so inhibited by caffeine that their G₁ is extended to>14 hours or that they are no longer proliferating cells. IBMX-induced binucleate cells, by contrast, did enter S phase and many of them also divided. Though in IBMX-induced binucleate cells there was also a decrease in nuclear volume up to 1+10 hours, subsequently mean nuclear volume increased e.g. at 1+16 and 1+18 hours. Both caffeine and IBMX treatments resulted in decreases in mean volume of prophase nuclei of mononucleate cells; this is further evidence that both methylxanthines inhibit the macromolecular synthesis required to sustain nuclear growth. It also suggests that nuclear division can be initiated at considerably lower nuclear volumes than those of untreated cells. We suggest that caffeine may act as a mimic of the normal mechanism that regulates the switch from a proliferating to a non-proliferative condition.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. I. Asynchronous mitosis of two nuclei in a single cell

Coordination of karyokinesis of two nuclei in individual filamentous binucleate cells of the fern,Adiantum capillus-veneris was investigated. To induce binucleate cells, the protonemata were treated with caffeine, which is known as an inhibitor of plant cytokinesis, during the first synchronous division of cells that was induced by blue light (BL). The next synchronous division of cells in the resultant binucleate cells was analysed. In most cases, the two nuclei were associated with each other and were located in the apical region of the long protonemal cells (approximately 400–600 μm in length, 20 μm in width). In some cells, one nucleus was located in the apical region and the other was located in the middle of the cylinderical region. In such cells, karyokinesis of the apical nucleus preceded that of the basal nucleus, even though karyokinesis of associated nuclei progressed synchronously. Mitotic binucleate cells were centrifuged in order to gather two dissociated heterophasic nuclei. Progression of karyokinesis in the re-associated nuclei became coordinated within 1 h in most cells. These results suggest that mitosis-regulating factor(s) may diffuse to only limited distances inAdiantum protonemata.     

Origin of mono- and binucleate cells with heterochromatic chromosomes in the malpighian tubules of the european elm scale,Gossyparia spuria (Coccoidea: Homoptera)

Males of the European elm scale, Gossyparia spuria (Erioccoccidae) have two Malphigian tubules, each made up of mononucleate and binucleate cells. Both types of cells may contain heterochromatic (H) chromosomes which form an H body. The cells with H bodies (H cells) usually appeared singly anywhere along the tubule. However, when two or more H cells were present they tended to be closer to each other than would be expected by chance. The possible origin of this tendency is discussed. Following squashing, the nuclei of the binucleate cells were much larger than those of most other somatic cells, suggesting that they were highly endopolyploid. However, the H bodies of the cells of the tubules were of about the same size as those of the other cells. These observations suggested that the H chromosomes of the binucleate cells did not replicate while the euchromatic chromosomes of these cells replicated several times. The great majority of the nuclei of the H cells contained a single H body per nucleus. An analysis of the number of H bodies in binucleate cells indicated that when two H bodies were present in the same nucleus they usually did not fuse. Thus, they were believed also not to fuse in the mononucleate cells. Since almost all the mononucleate H cells had only a single H body (rather than 2) it was concluded that they did not originate from binucleate cells by nuclear fusion.     

Membrane dynamics during migration of placental cells through trophectodermal tight junctions in sheep and goats

Binucleate cells in ruminant trophectodermal epithelium are unique in that they form part of the tight junction as they migrate across it, maintaining the ionic barrier seal to the internal milieu of the fetus. Such participation imposes considerable constraints on the cell migration because membrane cannot flow through a tight junction. We report quantitative ultrastructural immunocytochemical evidence for vesicle membrane insertion into the binucleate cell plasmalemma which allows the cells to form a pseudopodium past the tight junction. This pseudopodium increases continuously in area by vesicle insertion and develops a close apposition to the plasmalemma of the fetomaternal syncytium which constitutes the fetomaternal boundary in the placenta of the sheep and goat. Enventually the apposed membranes of the binucleate cell pseudopodium and the syncytium fuse by vesiculation and the cytoplasm and nuclei of the binucleate cell merge into the fetomaternal syncytium. The binucleate cell plasmalemma remaining on the trophectodermal side of the tight junction is blebbed off into, and phagocytosed by, the uninucleate trophectodermal cells between which the binucleate cell passed. This process permits the delivery of the binucleate cell granules to the maternal side of the placenta but none of the fetal molecules expressed on the plasma membrane of the binucleate cells are exposed to potential maternal immunological rejection.     

The double nucleus of the sertoli cell in the lizard Anolis carolinensis

Over 90% of the Sertoli cells in the testes of adult lizards (Anolis carolinensis) are binucleate. The nuclei occur in closely associated pairs in the basal cytoplasm of the Sertoli cells that line the testis tubules. The two nuclei of a pair are of similar volume, and each usually contains one conspicuous rounded nucleolus. The average volume of individual nuclei varies from 367.8 mum(3) in spermatogenically active testes in March to 172.5 mum(3) in September, when testes are regressed. The irregular shape of the Sertoli nuclei is particularly pronounced during testicular regression. Until initiation of spermatogenesis in hatchling lizards, Sertoli cells have a single nucleus containing patches of hetcrochromatin. With the appearance of prophase stages of primary spermatocytes, a few paired Sertoli nuclei can be found, and the nuclei increasingly exhibit the homogeneous euchromatic nucleoplasm of the adult. The average volume of individual nuclei in lizards under 4 months of age is less than a third the volume of Sertoli nuclei in reproductivcly active adults. The appearance of binucleate cells at this time documents a doubling of the amount of desoxynucleic acid in Sertoli cells preparatory to their growth and expanded functions during spermatogenesis.     

The degree of coupling of nuclear rotation in binucleate 3T3 cells

In order to test the existence of mechanical coupling between the rotational movements of two adjacent nuclei, we prepared binucleate 3T3 cells and observed their nuclear movements by near infrared microscopy and recorded them with time-lapse video techniques. We found that 49 out of 110 (44%) of the selected binucleate cells expressed nuclear rotation. Rotation could occur in just one of the nuclei while the second nucleus remained stationary (31/110) or in both nuclei simultaneously (18/110). In almost all cases where both nuclei rotated simultaneously (15/110) they did so at different speeds and in opposite directions. The nuclei were observed to rotate in the same direction in only three of the examples. The results are consistent with a weak mechanical interaction between a rotating nucleus and its neighbor. Consistent with our previous observations in mononucleate cells, we did not find a characteristic position of the centrosphere or a special distribution of the microtubules or the intermediate filaments in binucleate cells with rotating nuclei. There was an absence of long, well-formed microfilament bundles beneath the nuclei during rotation, even in the local region beneath the rotating nucleus in those cells with one rotating and one stationary nucleus. Also consistent with observations of mononucleate cells, nuclear rotation was inhibited by treatment with colcemid, although the ability of the nuclei to rotate was eventually restored when the colcemid-containing medium was replaced with normal medium.     

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division.
In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.     

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

On the triggering of mitosis and the division cycle of polynucleate cells

In binucleate 2n-2n and 4n-4n, trinucleate 2n-4n-2n and tetra nucleate cells 2n-2n-2n-2n which had been experimentally induced by means of caffeine (0.1% in tap water) in root-tip cells of onion bulbs (Allium cepa) division cycle time increases sligthly (about 15%) when the DNA content increases from 2n to 8n chromosomes per cell. The interphase time is not significantly modified, whereas the mitosis time increases (about 50%) in the tetranucleate cells in relation to the diploid mononucleate cells.The unsynchronized initiation of prophase and the subsequent synchronization of the nuclei in the polynucleate cells suggest an inhibiting mechanism regulating initiation of the mitosis via cytoplasm.     

Changes in pancreatic acinar cell nuclear number and DNA content during aging in the rat

Pancreatic acinar cells from rats 5 to 658 days (94 weeks) of age were isolated by enzymatic dissociation and stained with the DNA specific fluorochrome Hoechst 33258. The nuclear DNA content and the incidence of binucleation were estimated in these cells. Total pancreatic weight, RNA, protein and DNA, and the incorporation of 3H-thymidine into pancreatic acinar cell DNA were also estimated in similar animals as measures of pancreatic growth. From 5 to 17 days after birth, 95% of the cells were mononucleate diploid and 5% were binucleate diploid; but during the period of rapid pancreatic growth over the following 39 days, acinar cells became increasingly binucleate. By 56 days after birth, 64% of cells were binucleate with a diploid DNA content per nucleus; and the incidence of binucleation then remained constant. At 28 days of age, 4% of mononucleate cells were tetraploid, increasing to 6% at 658 days of age. At this time 3% of binucleate cells contained dual tetraploid nuclei. There is thus a rapid development towards diploid binucleate acinar cells in the growing, postnatal pancreas; and in the adult pancreas a small proportion of these cells develop tetraploid nuclei.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

Cell division in caffeine-induced binucleate protonemal cells ofAdiantum. II. Formation of the preprophase band and cell division in centrifuged and non-centrifuged cells

Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the PPB is discussed.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Cell population heterogeneity in the inducibility of DNA synthesis in human diploid fibroblasts

The initiation of nuclear DNA synthesis has been studied in cytochalasin B (CB)-induced binucleate human diploid fibroblasts (WI-38 cells). Mitotic cells from different passage levels were rendered binucleate by a brief pulse of CB. The cells were then washed free of the drug, and DNA synthesis was studied by [3H]thymidine labeling. The results showed that, in a small percentage of binucleate cells, one nucleus was labeled (S phase) and the other nucleus was unlabeled (G1 phase). There was no significant difference in the percentage of these cells with increasing passage levels. The results of this study suggest that some WI-38 cells retire from the cell cycle at different passage levels, and thereby become refractory to inducers of nuclear DNA synthesis generated by sister cells in S phase.     

Predicted performance of single- versus multiple-slit flow systems.

Flow systems utilizing multiple orthogonal excitation slits have been proposed as a means of reducing some types of false alarms in prescreening systems for gynecologic cytology. Such false alarms include those caused by orientation-dependent events, such as passage of binucleate or overlapping cells through the measurement region with both nuclei entering the excitation slit simultaneously. This paper presents distributions of optimal projection angles for randomly oriented nuclei passing through one, two, and three slit excitation regions. The results are used to compute observed nuclear spacing of binucleate cells and to compare performance of one, two, and three slit systems in recognition of binucleate and overlapping cells.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Genetic control of mitosis. Premature beginning of telophase chromatin reorganization in cells of the Drosophila melanogaster strain with ff3 mutation

The effect of cell cycle mutation ff3 on chromosome segregation was studied on fixed cells of neural ganglia. The cell distributions by diameter of interphase nuclei and by distance between sister chromatid sets were compared at anaphase and telophase. In the control wild-type strain Lausenne, the cell distribution by distance between sister chromatids in anaphase was similar to their distribution by nuclear size. The mean distance between segregating chromatids at anaphase (lcp) coincided with the mean diameter of interphase nuclei (dcp) and was 8.3 microns. Cells passed to telophase when chromatids were at least 10 microns apart. The mutant ff3 strain differed from the control strain Lausenne in cell distribution by interphase nuclear diameter and distance between sister chromatids in anaphase; the mean nuclear diameter and mean distance between segregating chromatids similarly increased to 9.3 microns. A specific feature of mitosis in mutant strain ff3 was a premature beginning of telophase chromatin reorganization. This caused the occurrence of cells with abnormally short (less then the interphase nuclear diameter) distance between sister chromatid sets in telophase but not in anaphase, as if these cells had passed from anaphase to telophase prematurely, during the chromatid movement toward poles in anaphase A.