Polyploid Mitoses Research Articles

Abstract 140: Polyaneuploid prostate cancer cells induced via chemotherapy have predominantly large, single nuclei

Abstract While localized prostate cancer (PCa) has a high treatment success rate, over 30,000 individuals in the United States and 350,000 men globally die each year from therapy resistant, metastatic PCa. A thorough understanding of the factors contributing to PCa recurrence following development of therapy resistance is acutely needed in order to develop efficacious treatments and disease-management strategies.Our group and others have identified a unique minority population of physically enlarged cancer cells with polyaneuploid genomes, termed polyaneuploid cancer cells (PACCs), in the tumors of patients with metastatic prostate cancer and in other tumor types. PACCs are the result of a polyaneuploid transition that occurs when aneuploid cancer cells experience environmental or therapeutic stress. These aneuploid cancer cells then pause their cell cycle after whole genome doubling to survive the environmental insult. Cancer cells in the PACC state appear to be quiescent and have an increased capacity for invasion/motility, an ability to endure in stressful or novel environments, and a propensity to undergo depolyploidization to re-establish the cancer cell population. We hypothesize that PACCs are critical players in prostate cancer therapy resistance and recurrence. We seek to better understand the life cycle of the PACC state in PCa in order to inform the design of new therapeutic strategies that prevent disease recurrence.One important part of understanding how cells enter and exit that PACC state is to characterize the features of the PACC nuclei compared to “typical” PCa cells. Examining the distribution of DNA content and the size of individual nuclei allows us to infer the underlying mechanisms PCa cells use to enter the PACC state, such as cell fusion, endomitosis, endocycling, or mitotic slippage. Nuclear structure can further indicate what types of depolyploidization are possible, including neosis, polyploid mitosis, or uneven cell division. Thus, we developed a robust method to isolate nuclei from PACCs and used flow cytometry and immunofluorescence imaging to ascertain the nuclear content and size from whole PACC cells as well as the isolated nuclei. Analysis of nuclear features from both whole cells and isolated nuclei is necessary to resolve truly multinucleated cells from those that contain a single polylobulated nucleus.Our preliminary data suggests that PCa cells in the PACC state tend to have a large polyploid single nucleus more frequently than multiple smaller, single nuclei, though both were observed. This is in contrast with the opinion of many in the field, who believe that PACCs (and other variations of giant polyploid cancer cells) only exist in a multinucleated state. Continuation of these studies in various cancer cell lines and at sequential timepoints in the PACC state will deepen our understanding of how cancer cells evade systemic treatment and cause disease recurrence. Citation Format: Anna Gonye, Chi-Ju Kim, Kenneth Pienta, Sarah Amend. Polyaneuploid prostate cancer cells induced via chemotherapy have predominantly large, single nuclei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 140.

Polyploid mitosis and depolyploidization promote chromosomal instability and tumor progression in a Notch-induced tumor model.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

The effects of biological and life-style factors on baseline frequencies of chromosome aberrations in human lymphocytes

Objective: This study investigated the influence of sex and ageing on chromosomal damage and the role of life-style habits on the frequency of chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) of healthy Bosnian subjects. Materials and Methods: Peripheral blood samples were obtained from 100 healthy, unrelated individuals in Bosnia and Herzegovina during 2010 and 2011. Chromosome preparations were made by dropping and air drying and slides were stained with 10% Giemsa solution (pH 6.8). The cytogenetic analysis was carried out in a cytogenetic laboratory in the Department of Biology of the Faculty of Science in Sarajevo. The category of total structural CAs was sub classified as chromosome-type aberrations (CSAs) and chromatid-type aberrations (CTAs) while the category of total numerical CAs was sub classified as aneuploid and polyploid mitoses. All statistical analyses were carried out using Microsoft Excel 2010 (Microsoft Corporation) and the Windows Kwikstat Winks SDA 7.0.2 statistical software package (Texa Soft Cedar Hill, Texas). Results: Cytogenetic analysis revealed the average number of structural CAs was 2.84 and of numerical CAs was 9.56. There was a significant increase in the frequency of chromosome-type aberrations (1.92) compared with chromatid-type aberrations (CTAs) (0.92) and a significant increase in the frequency of aneuploid (8.83) compared with polyploid (0.73) mitoses. Significant positive correlations between age and CTAs in human PBLs were also demonstrated. Additional statistical analysis showed that ageing increase number of numerical CAs in lymphocytes of drinkers. The frequency of structural CAs of females exposed to radiation was significantly greater than in males. Analysis indicates the presence of a positive association between CAs and smoking in younger subjects but a negative correlation between aberrant cells frequencies and alcohol in older drinkers. Conclusion: The results of the study support the conclusion that sex and age, together with life-style habits of individuals as confounding factors can affect spontaneous frequency of CAs especially to CTAs in PBLs that are a biomarker of cancer risk.

P53 regulates a mitotic transcription program and determines ploidy in normal mouse liver

Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the cell cycle, and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53(-/-) hepatocytes exhibited early entry into the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild-type (WT) and p53(-/-) hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2, and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene-specific manner. Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver.

Error-prone polyploid mitosis during normal Drosophila development

Endopolyploidy arises during normal development in many species when cells undergo endocycles-variant cell cycles in which DNA replicates but daughter cells do not form. Normally, polyploid cells do not divide mitotically after initiating endocycles; hence, little is known about their mitotic competence. However, polyploid cells are found in many tumors, and the enhanced chromosomal instability of polyploid cells in culture suggests that such cells contribute to tumor aneuploidy. Here, we describe a novel polyploid Drosophila cell type that undergoes normal mitotic cycles as part of a remodeling process that forms the adult rectal papillae. Similar polyploid mitotic divisions, but not depolyploidizing divisions, were observed during adult ileum development in the mosquito Culex pipiens. Extended anaphases, chromosome bridges, and lagging chromosomes were frequent during these polyploid divisions, despite normal expression of cell cycle regulators. Our results show that the switch to endocycles during development is not irreversible, but argue that the polyploid mitotic cycle is inherently error-prone, and that polyploid mitoses may help destabilize the cancer genome.

Chromosome pulverization in blood diseases.

Chromosome pulverization (premature chromosome condensation, PCC) was studied in bone marrow cells of healthy subjects and of patients suspected of having a blood disease. Cytogenetic preparations in 250 patients were made by the so-called direct method and in 33 subjects by the Harlequin technique (culture in 5-bromodeoxyuridine and staining with the fluorescent agent Hoechst 22358 and Giemsa (FPG)). By the direct technique, only 4 patients were found to have PCC: 1 untreated child with acute lymphocytic leukaemia (ALL), 2 treated children (one with ALL and the other with malignant lymphoma), and 1 adult who had acute non-lymphocytic leukaemia (ANLL) with megaloblastoid erythroid maturation. By the Harlequin technique another 4 patients, with megaloblastic anaemia, were shown to have PCC. However, neither the controls nor the patients with leukaemia had PCC by this technique. The patients who had PCC also had a high frequency of multinucleate cells in their bone marrow smears, or polyploid mitoses in the cytogenetic preparations. Thus, we believe that the abnormalities mentioned above cause nuclear asynchrony, which is generally regarded as the main reason for PCC.

Polyploid mitoses in human bone-marrow cells.

Polyploid mitoses in human bone-marrow cells.

Cytogenetic investigations from lymphocyte cultures of patients with probable Alzheimer's disease.

Chromosome investigations were carried out on lymphocyte cultures of 21 patients with probable Alzheimer's disease in comparison to an age-matched control group of 11 healthy subjects. Different cytogenetic parameters were analyzed: sister chromatid exchanges, polyploid mitoses, mitotic activity and secondary chromosomal aberrations such as gaps, breaks and exchanges. Only the average rate of sister chromatic exchanges was slightly decreased in patients (8.4 +/- 1.7; control group: 10.4 +/- 2.1). There were interindividual differences of values for each of the cytogenetic parameters analyzed, but these were not related to the onset, duration or severity of dementia.

Long-term callus cultures of diploid barley (Hordeum vulgare). II. Effect of auxins on chromosomal status of cultures and regeneration of plants

Chromosomal abnormalities increased with time on the relatively high concentrations (2.0mg/l or more) of 2,4-D. Prolonged exposures of cell cultures to high concentrations of 2,4-D significantly increased the percent polyploid mitoses and were also associated with increased abnormalities of the spindle apparatus. Cultures maintained in the presence of NAA had a large number of cells with structural alterations of the chromosomes. The best embryogenic callus production (those maintained on 0.1 and 1.0 mg/l 2.4-D) occurred in cultures with least detectable genetic variation. Plants could not be regenerated from cultures that had been on the maintenance media for seven months or more. The loss of regeneration potential was uniform across the concentrations and auxins tested, and only correlated with time. This loss could not be associated directly with detectable genetic variation.

1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells

We have investigated the cytogenetic effect of 1,6-dinitropyrene (1,6-DNP) in Chinese hamster V79 cells. The chemical caused a dose-dependent increase in the incidence of initial and full C-mitoses, polyploid mitoses, ana-telophases with lagging chromosomes, non-disjunction and multipolar configurations, in a range of 0.05–5 μM. These findings indicate that 1,6-DNP interferes with the functioning of the spindle apparatus in V79 cells. Early signs of spindle disturbances were seen at 1,6-DNP concentrations which only moderately reduced cell growth and division. Analysis of structural chromosomal aberrations revealed the appearance of chromatid-type aberrations with open breaks and exchanges accompanied by gaps. The results indicate that 1,6-DNP is both a spindle-disturbing and a clastogenic agent in V79 cells.

Behaviour of chromosomes in potato leaf tissue cultured in vitro as studied by BrdC-Giemsa labelling

Cells of leaf explants of a monohaploid potato (Solanum tuberosum) were stimulated to mitosis on a medium with 5-bromodeoxycytidine during a period of 7 days. The cells cycled with mono- or diplochromosomes which showed differential staining of the sister chromatids and sister chromatid exchanges by the fluorescent plus Giemsa technique after two rounds of BrdC incorporation. Through the staining pattern the course of the first three cell cycles could be traced and the duration of the cycles estimated. Polyploidisation was enhanced by selective stimulation of polyploid cells and by endoreduplication of G2-phase cells. The percentage of polyploid mitoses increased from 10 to 70.

Near-Tetraploid Clones in Acute Leukemia

Near-tetraploid clones were observed in bone marrow (BM) samples from two patients with acute leukemia. One case had a hypotetraploid clone (model chromosome range, 88-90) characterized by two apparently identical 8:21 translocations and loss of both Y chromosomes as well as by other changes. As in aneuploid patients with only a single 8:21 translocation, the cytologic features in this case were characteristic of acute myeloid leukemia with maturation. The marrow of the second patient contained immature leukemic blasts indicative of acute myeloid leukemia. The abnormal clone in this patient contained 93 chromosomes with an extra small marker. In this case we examined BM preparations that were made by several different cytogenetic methods: "direct" and 24-hr culture (with or without methotrexate synchronization). We scanned 100 consecutive mitotic cells from each of the three preparations and found almost no variation in the incidence of abnormal metaphase cells. However, in this particular case, we noted some disparity between preparations with respect to the quality of diploid mitoses versus that of near-tetraploid mitoses. This finding is discussed with near-tetraploid mitoses. This finding is discussed with regard to bias in selecting metaphase cells for banding analysis in acute leukemia in general. In both of our patients there was a close correlation between the incidence of polyploid mitoses observed in cytogenetic preparations and the incidence of bizarre blast cells found in BM smears on the same date.

Near-tetraploid clones in acute leukemia

Near-tetraploid clones were observed in bone marrow (BM) samples from two patients with acute leukemia. One case had a hypotetraploid clone (model chromosome range, 88–90) characterized by two apparently identical 8:21 translocations and loss of both Y chromosomes as well as by other changes. As in aneuploid patients with only a single 8:21 translocation, the cytologic features in this case were characteristic of acute myeloid leukemia with maturation. The marrow of the second patient contained immature leukemic blasts indicative of acute myeloid leukemia. The abnormal clone in this patient contained 93 chromosomes with an extra small marker. In this case we examined BM preparations that were made by several different cytogenetic methods: “direct” and 24-hr culture (with or without methotrexate synchronization). We scanned 100 consecutive mitotic cells from each of the three preparations and found almost no variation in the incidence of abnormal metaphase cells. However, in this particular case, we noted some disparity between preparations with respect to the quality of diploid mitoses versus that of near-tetraploid mitoses. This finding is discussed with near-tetraploid mitoses. This finding is discussed with regard to bias in selecting metaphase cells for banding analysis in acute leukemia in general. In both of our patients there was a close correlation between the incidence of polyploid mitoses observed in cytogenetic preparations and the incidence of bizarre blast cells found in BM smears on the same date.

A comparative karyological and cytophotometric study of normal and wounded stem tissue of the pea (Pisum sativum L.)

The garden pea (Pisum sativum L.) is subject to crown gall tumors. In an attempt to understand the process of initiation of these tumors karyological studies of wounded stem tissue have been made and the results compared with those of karyological and cytophotometric studies of unwounded stem tissue.Wounding induced increased mitotic activity with polyploid mitoses becoming relatively more frequent. These polyploid cells are more likely to have been present in the tissue at the time of wounding rather than induced by the wounding. Mitoses were found commonly in vascular and pith tissue but not in the cortex. The region of active cell division extended about 500μm from the wound surface. The wound-induced mitotic activity continued for approximately three weeks. The level of mitotic activity and the relative frequency of polyploid mitoses was similar to that found earlier in crown gall tumor tissue, the difference being that the tumor tissue remained mitotically active for approximately three months.

Plant Regeneration in Wheat Tissue Culture

Cytologital analysis of 65 plants regenerated from callus tissue of a common wheat (T. aestivum) in R 1 and R 2 generations revealed their mixoploid structure. Dominance of haploid cells and cells with near haploid chromosome sets in majority of these plants was observed. Aneuploid cells occured in the plants with significant frequency (from 9.9 to 84.4%). Diploid cells in the plants of 5 lines were very rare and in some cases were absent at all. The frequency of polyploid mitoses was low (up to 8.6%). Young plants regenerated from calli differed from seed plants by the growth rate, height and the number of tillers at early stages of development. Later on these differences disappeared.

Origin of the early megakaryopoietic progenitor cell and further polyploidization in the thrombopoietic cell series. Phase-contrast observations of human bone marrow.

Continuous observation of living cells by phase-contrast time lapse microcinematography revealed different origins of multinuclear cells and cells with lobed nuclei in the hematopoietic progenitor cell series, which must be classified as megakaryoblasts, the determined progenitor cells of the megakaryopoietic series. Hematopoietic progenitor cells with single nuclei of nuclear diameter 5-13 microns, the promegakaryoblasts, may become polyploid by various means: by postmitotic cytoplasmic fusion, by consecutive nuclear fusion, by endomitosis, or by nuclear amitosis or lobe formation presumably after endoreduplication and nuclear growth. Including amitotic nuclear division in the mitotic rate of normal progenitor cells nearly the half of all these cells in G2 phase (0.4) produce in vitro octoploid megakaryoblasts, respectively osteoclasts. Thrombopoietin-rich sera in the medium enhance the mitotic index of the hematopoietic progenitor cell line from 0.1 to 0.25, but do not change the proportion of polyploid mitoses.

Growth of mouse megakaryocyte colonies in vitro.

Mouse bone marrow and spleen cells formed pure or mixed colonies of up to 80 megakaryocytes in agar cultures after stimulation by medium conditioned by activated mouse lymphoid cells. Megakaryocytes were identified on the basis of their morphology, polyploid mitoses and DNA content, and high cytoplasmic content of acetylcholinesterase. Megakaryocyte colony-forming cells were relatively small with a peak sedimentation velocity of 4.2 mm/hr. Spleen, lymph node, and thymus cells produced the factor stimulating megakaryocyte proliferation after culture in medium containing 2-mercaptoethanol, with or without added mitogens or allogeneic spleen cells. Peak activity in conditioning medium was associated with the small lymphocyte fractions in mouse spleen.

The human leukocyte test system

In human leukocyte cultures st up with TC medium 199,DNA synthesis and mitotic indices were analysed by means of 3H-thymidine autoradiography and cell counting. DNA synthesis starts at around 28 hrs. The frequencies of labelled cells rise slowly and reach a maximum of around 24%. The first mitoses appear at around 38 hrs but up to 49 hrs only very few mitoses can be seen. After that time the mitotic indices rise and reach values of up to 11% cultivation in the presence of BudR for 72 hrs and staining with Hoechst 33258 stain revealed that first, second and third mitoses occur together in the cultures at this time. Irradiation of whole blood and cultivation for 72 hrs leads to mitoses containing dicentric and ring chromosomes with and without fragments, to interphases with micronuclei, to premature chromosome condensations (PCC) and to polyploid mitoses indicating that at this time first and further mitoses are present.

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

AbstractDNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with 3H‐thymidine in slides in which mitoses had been counted and analyzed before autoradiography.The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two.The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.

Abscisic acid inhibition of root nodule initiation in Pisum sativum

The effect of exogenous abscisic acid (ABA) on root nodule formation in Pisum sativum cv. Alaska was examined. ABA supplied to the roots at 1.9×10(-6)M reduced the number of nodules/plant 61% without affecting root or shoot growth. The first noticeable inhibition of nodulation occurred at 3.8×10(-7)M ABA. ABA at a concentration of 1.9×10(-6)M inhibited neither root hair formation nor infection of root hairs by Rhizobium leguminosarum. Similar numbers of infection threads penetrated the cortex in both control and treated plants. ABA concentrations of 3.8×10(-6)M had no effect on the doubling time or maximum density of R. leguminosarum in pure cultures. Normal nodule formation involves a polyploid cortical proliferation. This response to rhizobial infection can be imitated by culturing 1-mm pea-root segments on a medium containing 4.7×10(-6)M kinetin. Under these conditions a highly significant reduction in the number of polyploid mitoses after 72 h is produced by 3.8×10(-8)M ABA. A maximum inhibition of 68% was found with 3.8×10(-6)M ABA. A similar range of ABA concentrations also inhibited the cytokinin-induced cell division in soybean callus. It is concluded that ABA reduces the number of root nodules/plant by inhibiting the cortical cell divisions required for root nodule formation.