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.     

CHRONOCRISIS: When Cell Cycle Asynchrony Generates DNA Damage in Polyploid Cells

Polyploid cells contain multiple copies of all chromosomes. Polyploidization can be developmentally programmed to sustain tissue barrier function or to increase metabolic potential and cell size. Programmed polyploidy is normally associated with terminal differentiation and poor proliferation capacity. Conversely, non-programmed polyploidy can give rise to cells that retain the ability to proliferate. This can fuel rapid genome rearrangements and lead to diseases like cancer. Here, the mechanisms that generate polyploidy are reviewed and the possible challenges upon polyploid cell division are discussed. The discussion is framed around a recent study showing that asynchronous cell cycle progression (an event that is named “chronocrisis”) of different nuclei from a polyploid cell can generate DNA damage at mitotic entry. The potential mechanisms explaining how mitosis in non-programmed polyploid cells can generate abnormal karyotypes and genetic instability are highlighted.     

Tumorigenic polyploid cells contain elevated ROS and ARE selectively targeted by antioxidant treatment

Polyploidy has been linked to tumorigenicity mainly due to the chromosomal aberrations. Elevated reactive oxygen species (ROS) generation, on the other hand, has also been associated with oncogenic transformation in most cancer cells. However, a possible link between ploidy and ROS is largely unexplored. Here we have examined the role of ROS in the tumorigenicity of polyploid cells. We show that polyploid prostate and mammary epithelial cells contain higher levels of ROS due to their higher mitochondrial contents. ROS levels and mitochondrial mass are also higher in dihydrocytochalasin B (DCB)-induced polyploid cells, suggesting that higher levels of ROS observed in polyploid cell can occur due to cytokinesis failure. Interestingly, polyploid cells were more sensitive to the inhibitory effect of the antioxidant, N-Acetyl-L-cysteine (NAC), than control diploid cells. Treatment of polyploid/diploid cells with NAC led to the selective elimination of polyploid cells over time and abrogated the tumorigenicity of polyploid cells. This effect was partially mediated via the Akt signaling pathway. We next explored a possible role for ROS in promoting chromosomal instability by analyzing the effects of ROS on the mitotic stage of the cell cycle. Enhancing ROS levels by treating cells with hydrogen peroxide delayed not only entry into and but also exit from mitosis. Furthermore, increasing ROS levels significantly increased taxol resistance. Our results indicated that increased ROS in polyploid cells can contribute to tumorigenicity and highlight the therapeutic potential of antioxidants by selectively targeting the tumorigenic polyploid cells and by reversing taxol resistance.     

Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigenin-labelled probes

Summary DNA-DNA in situ hybridization, with two digoxigenin-labelled, chromosome-specific DNA probes, was used to determine the number of copies of a given chromosome in interphase nuclei and so identify putatively polyploid nuclei in histological sections of several mouse tissues. One hybridization site per diploid genome was expected for tissues with hemizygous markers: male mice hybridized with a Y chromosome probe (pY353/B) or hemizygous transgenic mice hybridized with a -globin probe (pM02). Nuclei with more than one hybridization site were considered putative polyploids. Three groups of experiments were undertaken: (1) evaluation of the method, using mouse liver sections; (2) studies of tissues already known to contain polyploid nuclei, and (3) studies that resulted in the discovery that the mouse ovary contains polyploid nuclei. First, control studies showed that the ability to detect the target DNA sequences was affected by section thickness. Studies of nuclear ploidy in the developing mouse liver revealed a pattern similar to that established by previous studies using DNA content as a criterion for ploidy. At birth, only about 5% of the liver nuclei were polyploid; this increased to 10–15% by 10–20 days and was followed by a sharp increase in the frequency of tetraploid nuclei between 20 and 40 days (to about 35%) and a more gradual increase in higher order polyploid nuclei. Secondly, this technique was used to confirm that polyploid (mostly tetraploid) nuclei were present in the bladder epithelium, heart, uterine decidua and placental trophoblast. Higher order polyploidy was seen in large bone marrow cells (megakaryocytes) but not in the even larger trophoblast giant cells of the placenta, thus confirming previous claims that these cells are polytene rather than polyploid. Thirdly, putatively tetraploid nuclei were found in the ovarian follicle and corpus luteum. As far as we are aware, this is the first time polyploid nuclei have been reported for the mouse ovary.     

Growth of cardiac muscle cells during rat adaptation to altitude hypoxia

The weight of the right heart ventricle in 1.5-month-old rats kept after birth in the mountains of 3400 m altitude is higher and its muscle cell cytoplasm mass is much larger compared to those in 1.5-month-old animals raised at 800 m altitude. The hypertrophy of cells is not due to their polyploidization. Only a small increase in the relative number of polyploid cells takes place under high altitude hypoxia. The weight of the right ventricle and myocyte mass in 3-month-old rats kept 1.5-3 months after the birth at 3400 m altitude also increases, although this augmentation is significantly less than in the animals grown in the mountains for 1.5 months immediately after the birth. The myocyte ploidy of adult animals adapted to hypoxia does not essentially differ from that of 1.5- and 3-month-old control rats: about 80 per cent of these cells are polyploid. Thus, the growth of cardiac myocytes under the heart hyperfunction in the case of high altitude hypoxia proceeds mainly on the ground of the stable polyploid genome, as well as normal ontogenetic growth of these cells.     

Induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene

The induction of chromosome aberrations in lymphocytes of mice after subchronic exposure to benzene was investigated. 4 groups of 5 Swiss (ICR) male mice were given orally a solution of benzene every day for 14 days except days 5 and 10. The daily doses were 0, 36.6, 73.2 and 146.4 mg/kg. Mice were sacrificed on day 15, lymphocytes were obtained by perfusion of the spleen and the cells were cultured in RPMI 1640 medium. After 48 h of culture, cells were harvested for cytogenetic analysis. A significant dose-dependent increase in the frequency of cells with chromatid aberrations were found (p less than 0.001). A significant increase in polyploid cells were also observed (p less than or equal to 0.05). This study represents the first report on the induction of chromosome aberrations and polyploid cells in lymphocytes of mice after subchronic exposure to benzene. Such dual activity of benzene suggests that benzene may be responsible for more human health problems than currently estimated.     

Bovine embryos contain a higher proportion of polyploid cells in the trophectoderm than in the embryonic disc

The frequency of polyploid cells in the embryonic disc (ED) and in the trophectoderm (TE) was assessed in 50 in vitro produced bovine embryos fixed at days 7-8 post insemination (pi) and in 20 in vitro produced embryos that were transferred to uteri of recipients at day 7 and then recovered and fixed at day 12 pi. Separation of TE and ED cells was obtained by microdissection and the frequency of polyploid cells was determined by interphase cytogenetic analysis using fluorescence in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes. The results show that 96% of day 7 embryos contain polyploid cells in the TE, whereas only 58% contain polyploid cells in the ED. In day 12 embryos 85% of TE and 40% of ED preparations contain polyploid cells. Statistical analysis revealed that the frequency of polyploid cells was significantly higher in the TE than in the ED in embryos containing less than 25% polyploid cells (n = 65). The few embryos (n = 5), which contained more than 25% polyploid cells, did not show this difference. Further, it was revealed that the level of polyploidy on day 7-8 was significantly higher than on day 12, both in the TE (two-fold) and in the ED (seven-fold).     

Changes in Deoxyribonucleic Acid Synthesis Regulation in Chinese Hamster Cells Infected with Simian Virus 40

Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G(1), G(2), or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.     

Manifestation of malignancy in somatic hybrids obtained by the cell fusion of 2 tumor lines

Malignancy of 6 independent hybrid clones derived from fusion of two tumor cell lines of Djungarian hamster, which had been transformed with SV40 virus, was studied. In most of the hybrid clones, suppression of the ability to grow progressively in vivo and the increase in the latent period of tumor occurrence were observed. These data bear witness to suggestion about the existence of different genetic alterations in these tumor cells. Suppression of malignancy does not depend on the genome mutations leading to cell polyploidization, since no decrease in tumorigenicity was found in polyploid cell clones of the high tumor cell line. These polyploid cells can actively form colonies in the soft agar medium.     

Kinetics of endomitosis in primary murine megakaryocytes

Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.     

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. I. Dynamics of polyploidization

Polyploidization of the extravillous trophoblast (EVT) cells at different stages of differentiation and invasion into the uterine wall in human placenta has been studied. An increase in the ploidy level of EVT cells in the course of their differentiation within cell columns (CC) was shown. Stem cells were mainly diploid (86.2%); incidence of polyploid nuclei of highly proliferative cells of the proximal part of CC increased progressively. In the distal part of CC, where EVT cells did not divide mitotically, polyploid cells prevailed, with 58.0 and 3.5% nuclei being 4c and 8c, respectively. The highest percentage of polyploid cells was found in the population of EVT cells attached directly to the surface of the decidualized endometrium: percentage of tetraploid cells turned out to be 74.7% and the share of octaploid nuclei rose up to 4.9%; however, there appeared a few (0.3%) 16c cells. The majority of EVT cells invading the decidualized endometrium were polyploid, the share of octaploid and hexadecaploid cells rose up to 9.7 and 1.4%, respectively. On the other hand, the percentage of diploid cells also increased up to 29.2% as compared to EVT cells attached to decidua (20.0%). The same tendency proved to be even stronger in myometrium: the share of diploid EVT cells increased up to 46.0%, a prominent amount of tetraploid (45.1%) and highly polyploid (8c and 16c) cells retained in the EVT cell population (7.4 and 1.1%, respectively). Immunohistochemical staining of Ki-67 protein (MIB1), which labels cells held in the cell cycle, showed a high incidence of MIB1-positive stem cells (93.7%) and the EVT cells of the proximal part of CC (85.5%) characterized by high mitotic activity. A lower MIB1-positivity (43.2%) was found in the distal part of CC, whereas invasive EVT cells showed no MIB1-labeling. The presence of MIB1-positive nuclei in the distal part of CCs in the absence of mitoses, taken together with data on polyploidization of these cells, indicates their switch to the endoreduplication cycle. As a whole, the data obtained evidence that differentiation of EVT cells of the invasive pathway is accompanied by polyploidization. However, in a population of trophoblast cells capable of most profound invasion (up to myometrium), the proportion of diploid cells rose. These results suggest that the human cytotrophoblast invasion into the uterine wall requires an optimum, not the highest, ploidy level, whereas highly polyploid cells may form a subpopulation at the border between the maternal and fetal parts of placenta.     

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.     

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4?days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2?weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3?days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.     

CELL POPULATION KINETICS IN CALLUS TISSUES OF CULTURED PEA ROOT SEGMENTS

Two callus tissues, one composed of diploid and the other of a mixture of diploid and polyploid cells, were derived by culturing 1-mm pea root segments; the mixed callus tissues were obtained by incorporation of kinetin in the culture medium. The callus tissues were used to determine (a) if cell proliferation was altered with the change in cell constituents of a callus; (b) the rate at which polyploid cells increased after kinetin stimulation; (c) the nature of the mitotic cycle in the diploid and mixed polyploid callus tissues; and (d) if the mitotic cycle changed as the tissue aged. Histological, cytological, radioisotope, and radioautographic analyses were made on callus tissue ranging from 1 to 4 days old. The results indicated that gross morphological changes were associated with the anatomical location of the proliferative cells. They showed that the percentage of polyploid division figures after stimulation by kinetin increased rapidly during the first 6 to 7 days in culture and then continued to increase at a much reduced rate. Cell counts revealed that cell proliferation in the mixed callus tissue was initially delayed when compared with the diploid tissue, but that after the delay was overcome cell number increased in each in similar manner. Analysis of the number of DNA-synthesizing cells showed that their percentage was highest during the first 2 days of culture and then leveled off at a value of about 10%. Mitotic cycle analysis indicated that it could be accurately measured only in the younger diploid callus tissues and that it increased in variability with increased age.     

Pluripotency of a polyploid H1 (ES) cell system without leukaemia inhibitory factor

Objectives: Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)‐cell system was prepared. Materials and methods: Diploid, tetraploid, pentaploid, hexaploid, octaploid and decaploid H1 (ES) cells (2H1, 4H1, 5H1, 6H1, 8H1 and 10H1 cells, respectively) were cultured for about 460 days in L15F10 medium without leukaemia inhibitory factor (LIF). The cells cultured under LIF‐free conditions were denoted as 2H1(?), 4H1(?), 5H1(?), 6H1(?), 8H1(?) and 10H1(?) cells, respectively. Pluripotency and gene expression were examined. Results: Ploidy alteration of H1(?) cells was similar to that of H1 cells. The polyploid H1(?) cells showed positive activity of alkaline phosphatase, suggesting that they maintained pluripotency in vitro without LIF. The polyploid H1(?) cells formed teratocarcinomas in mouse abdomen, suggesting they could differentiate in mouse abdomen in vivo. 2H1, 4H1 and polyploid H1(?) cells expressed nanog, oct3/4 and sox2 genes, suggesting that they fulfilled the criteria of ES cells. Nanog gene was significantly over‐expressed in 4H1 and polyploid H1(?) cells, suggesting that overexpression of nanog gene was a characteristic of polyploid H1 cells. Conclusion: Polyploid H1 (ES) cells retained pluripotency in vitro, without LIF with nanog over‐expression.     

Cytogenetic effects of the addition of leukocytes and blood sera on human fibroblasts exposed to measles virus or to streptolysin-O

Infection with measles virus and also introduction of streptolysin-O induced a significant increase in the level of cells with cytogenetic disturbances in the culture of human fibroblasts (HF). A decrease to intact condition of the number of HF with aneuploid and polyploid sets of chromosomes was observed after the introduction of non-immune autologous T-lymphocytes into the cultures. Immune homologous T-lymphocytes, unlike non-immune autologous T-lymphocytes, eliminated cells with structural disturbances of the chromosomes from the culture, but did not influence the level of aneuploid and polyploid cells. The ability of immune T-lymphocytes to exert antimutagenic effect can obviously be explained by their cytolytic action on virus-infected cells. As for non-immune autologous T-lymphocytes, two ways are equally probable: T-lymphocytes eliminate HF with virus antigens on their surface, or T-lymphocytes determine and destroy fibroblasts with changed cell surface developing as a result of cytogenetic disturbances induced by infectious factors. Specificity of the cytolytic reaction of T-lymphocytes concerning cells with some types of cytogenetic disturbances has been demonstrated.     

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.     

Effects of diethylstilboestrol-dipropionate on SCEs, micronuclei, cytotoxicity, aneuploidy and cell proliferation in maternal and foetal mouse cells treated in vivo

The effect of diethylstilboestrol-dipropionate on the frequency of SCEs and micronuclei, cytotoxicity, aneuploidy and cell proliferation rates of foetal liver and maternal bone marrow cells following exposure of pregnant mice was measured. An increase in the number of aneuploid and polyploid cells was observed in both tissues. There was no effect on micronuclei frequency, SCE frequency, or cell proliferation rate.     

Change n the ploidy classes of left ventricular myocytes in rats after ligation of the left coronary artery

Heterogeneous response of cardiomyocytes in left atria of rats to ligation of the left coronary artery was observed. Ploidy may greatly increase reaching 32 c per cell. More than 90% of myocytes are normally mononuclear diploid cells, while after the ligation the number of such cells diminishes to 10%. The majority of cells become polyploid: above 50% of cells are 2 X 2 c ploid, about 30% of cells are 4 X 2 c ploid and a great number of cells are highly ploid.     

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.