Endomitotic Process Research Articles

Defective endomitosis during megakaryopoiesis leads to thrombocytopenia in Fanca−/− mice

AbstractFanconi anemia (FA) is an inherited chromosomal instability syndrome that is characterized by progressive bone marrow failure. One of the main causes of morbidity and mortality in FA is a bleeding tendency, resulting from low platelet counts. Platelets are the final products of megakaryocyte (MK) maturation. Here, we describe a previously unappreciated role of Fanconi anemia group A protein (Fanca) during the endomitotic process of MK differentiation. Fanca deficiency leads to the accumulation of MKs with low nuclear ploidy and to decreased platelet production. We show, for the first time, that Fanca−/− mice are characterized by limited number and proliferative capacity of MK progenitors. Defective megakaryopoiesis of Fanca−/− cells is associated with the formation of nucleoplasmic bridges and increased chromosomal instability, indicating that inaccurate endoreplication and karyokinesis occur during MK polyploidization. Sustained DNA damage forces Fanca−/− MKs to enter a senescence-like state. Furthermore, inhibition of the Rho-associated kinase, a regulator of cytokinesis, improves the polyploidization of Fanca−/− MKs but greatly increases their genomic instability and diminishes their differentiation potential, supporting the notion that accumulation of DNA damage through endomitotic cycles affects MK maturation. Our study indicates that Fanca expression during endomitosis is crucial for normal megakaryopoiesis and platelet production.

Presence of a defect in karyokinesis during megakaryocyte endomitosis

Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, a process corresponding to a late failure of cytokinesis with a backward movement of the daughter cells. Generally, a pure defect in cytokinesis produces a multinucleated cell, but megakaryocytes are characterized by a single polylobulated nucleus with a 2N ploidy. Here, we show the existence of a defect in karyokinesis during the endomitotic process. From late telophase until the reversal of cytokinesis, some dipolar mitosis/endomitosis and most multipolar endomitosis present a thin DNA link between the segregated chromosomes surrounded by an incomplete nuclear membrane formation, which implies that sister chromatid separation is not complete. This observation may explain why polyploid megakaryocytes display a single polylobulated nucleus along with an increase in ploidy.

Abstract 2022: P53-dependent thrombocytopenia induced by the histone deacetylase inhibitor S78454

Abstract S78454 (also called PCI-24781) is an orally bioavailable, hydroxamate-based pan-HDACi currently being tested in clinical trials in the US and EU. Like many other HDACi, this drug induces a reversible thrombocytopenia. This thrombocytopenia, which was associated with a decrease in either GATA1 transcriptional activity or the expression of proteins of the Rho GTPase family (RhoA, Cdc42 or Rac), remains poorly understood. Given that the S78454 plasma level is above 100nM in treated patients, we performed a dose-response analysis (from 10 to 100 nM) of the drug effects on CFU-MK growth and cell proliferation. CFU-MKs were generated by ex vivo culture of CD34-positive cells and their differentiation was dose-dependently decreased after either a 24-hour treatment or a continuous exposure to S78454. This effect was associated with a dose-dependent decrease in MK proliferation. When added at day 8 of the culture, at a time-point when MK have nearly finished their endomitotic process and are entering in the cytoplasmic maturation step, S78454 induced a dose-dependent decrease (ten- to- two fold at 100nM and 20nM, respectively) in proplatelet formation, even when secondarily removed from the culture medium. The decreased proliferation was associated with an increased apoptosis, as demonstrated by the presence of a sub-G1 peak after propidium iodide staining, Annexin V binding, and caspase-3 proteolysis. We did not observed any blockade in the TPO/MPL/JAK2 signaling since ERK, Stat3 and Stat5 phosphorylation remained unaffected. Interestingly, MK exposed to S78454 displayed an increase in γH2AX foci formation and a decrease in Rad51 expression, a major mediator of homologous recombination, indicating a defective repair of DNA double-strand breaks. Consequently, P53 became phosphorylated in MKs, and the expression of its target genes BAX and P21 was increased. Altogether, these results suggest that S78454-induced thrombocytopenia may be mediated by induction of apoptosis in MK due to DNA-double-strand breaks and p53 activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2022. doi:1538-7445.AM2012-2022

Aurora B is dispensable for megakaryocyte polyploidization, but contributes to the endomitotic process

AbstractPolyploidization of megakaryocytes (MKs), the platelet precursors, occurs by endomitosis, a mitotic process that fails at late stages of cytokinesis. Expression and function of Aurora B kinase during endomitosis remain controversial. Here, we report that Aurora B is normally expressed during the human MK endomitotic process. Aurora B localized normally in the midzone or midbody during anaphase and telophase in low ploidy megakaryocytes and in up to 16N rare endomitotic MKs was observed. Aurora B was also functional during cytokinesis as attested by phosphorylation of both its activation site and MgcRacGAP, its main substrate. However, despite its activation, Aurora B did not prevent furrow regression. Inhibition of Aurora B by AZD1152-HQPA decreased cell cycle entry both in 2N to 4N and polyploid MKs and induced apoptosis mainly in 2N to 4N cells. In both MK classes, AZD1152-HQPA induced p53 activation and retinoblastoma hypophosphorylation. Resistance of polyploid MKs to apoptosis correlated to a high BclxL level. Aurora B inhibition did not impair MK polyploidization but profoundly modified the endomitotic process by inducing a mis-segregation of chromosomes and a mitotic failure in anaphase. This indicates that Aurora B is dispensable for MK polyploidization but is necessary to achieve a normal endomitotic process.

Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling

AbstractMegakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

Isolation and culture of megakaryocyte precursors.

Megakaryocyte (MK) precursor cells correspond to a spectrum of cells extending from an early progenitor to the promegakaryoblast, a 2N cell that switches from a mitotic to an endomitotic process (). The MK progenitors express the CD34+ antigen and are either CD38– or CD38+, depending upon their maturation stage. The most primitive progenitors (BFU-MK and the mixed erythro-MK progenitors) are negative for CD38, while the others are CD38 positive (). Other differentiation markers, such as HLA-DR or AC133 (,), have also been used to study MK differentiation. In the adult, CD41 (platelet GPIIb) appears during differentiation and can be used to select a subset of MK progenitors that essentially correspond to the more mature MK progenitors and promegakaryoblasts. In contrast, CD41 is more widely expresssed on neonatal hematopoietic progenitors. CD42 has a slightly later expression than CD41 (5), although after the beginning of CD42 synthesis, expression levels of CD41 and CD42 are well correlated. Thus, MK differentiation may proceed by different stages: CD34+CD38–CD41–, CD34+CD38+CD41–, CD34+CD38+CD41+CD42–, CD34+CD41+CD42+, CD34–CD41+CD42+, CD34–CD41+++CD42+++ (Fig. 1). Open image in new window Fig. 1. Expression of different markers during MK differentiation. See introduction for further explanation.

Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

AbstractPloidy could be the key to understanding megakaryocyte (MK) biology and platelet production. Human CD34+ cells purified from umbilical cord blood (CB) and peripheral blood (PB) were investigated on their capability to give rise, in a serum-free medium containing thrombopoietin, to MKs and platelets. CB-MKs showed reduced polyploidization and platelet number compared with PB-MKs, but a similar membrane phenotype. Most CB-MKs showed a 2N content of DNA (∼80%) and only 2.6% had 8N, whereas 40% of the PB cells had 8N or more. Platelets were substantially released in PB culture from day 12; at day 14 the CB-derived MKs were able to release platelets although at a reduced level (∼35%), correlating with their reduced size. A direct correlation was demonstrated by sorting polyploid cells from PB-MKs and evaluating the platelets released in the supernatant. Furthermore, the study analyzed the expression and distribution of cyclin D3 and cyclin B1. Cyclin D3 protein was increased in PB in comparison to CB-MKs; in PB culture most cells rapidly became positive, whereas in CB-derived cells cyclin D3 expression was evident only from day 9 and in a reduced percentage. Cyclin B1 was essentially localized at the nuclear level in the CB and was expressed during the whole culture. In PB-MKs, at day 9, a reduction was observed, correlating with an advanced ploidy state. The data indicate the inability of the CB-MKs to progress in the endomitotic process and a direct correlation between DNA content and platelet production.

Development of muscle fibre nuclei in holometabolous insect larvae

The growth and development of the nuclear population from intersegmental dorsal longitudinal muscles (IDLM) of Bombyx mori and Pieris brassicae during larval development were studied. Using whole muscle mounts, examined with an image analysis system, we observed increases in muscle size but a constant number of fibres and concomitant increases in the number and size of fibre nuclei. The gradual growth of the nuclei throughout larval life, corresponding very probably to an increase in their DNA content, is unequal and results in heterogeneity of the nuclear population. This suggests periodic incorporation of myoblasts into the larval muscles; the nuclei arising from these myoblasts would therefore undergo endomitotic processes and grow polyploid. The possibility of somatic reduction divisions of the polyploid muscle nuclei is discussed.

The Role of DNA Synthesis During Hypocotyl Elongation in Light and Dark

Fluorodeoxyuridine, an inhibitor of DNA synthesis, did not affect germination and post-germinative growth in the aerial part of lettuce and Haplopappus gracilis seedlings when grown in the light. In the dark, however, elongation of the hypocotyl was inhibited by fluorodeoxyuridine, strikingly in lettuce and only slightly in Haplopappus gracilis. This could imply that the controlling mechanism of hypocotyl elongation is in some cases related to DNA synthesis, either because mitotic processes (often little taken into account in considering hypocotyl growth) are involved in the elongation of hypocotyls only when they are grown in the dark, or because DNA synthesis affects cell elongation directly, or through the production of a greater number of endopolyploid cells in the dark. Using mainly autoradiographic and cytofluorimetric methods, these possibilities were tested. Besides lettuce (Lactuca sativa L. var. Great Lakes) and H. gracilis (Nutt.) Gray, radish (Raphanus sativus L. var. Tondo rosso quarantino) and soybean (Soya hyspida Sieb. and Zucc. var. Tokyo) seedlings were also studied. Fluorodeoxyuridine drastically inhibits cell elongation only when it is preceded or accompanied by mitotic or endomitotic events. Need for DNA synthesis during hypocotyl elongation, as well as during early post-germinative growth, seems to be of particular importance when endomitotic processes are involved.

Cell fusion as a mechanism for the formation of giant cells (Langhans' type). Autoradiographic findings in autoimmune tubulo-interstitial nephritis of the rat.

The formation of multinuclear giant cells of the Langhans' type in tubulo-interstitial auto-immune nephritis in the rat has been investigated by means of autoradiography. While in the majority of giant cells all nuclei were radiolabeled, in a few both labeled and unlabeled nuclei were present. This latter finding represents strong evidence in favour of the hypothesis that giant cells do not form by endomitotic processes but rather through fusion of certain precursor cells. According to previous studies this precursor cell population consists mainly of epitheloid cells, i.e. modified monocytes.

The Phaseolus suspensor and its polytene chromosomes

The first division of the zygote of Phaseolus is an asymmetric one, thus establishing two cells with different developmental capacities. Whereas the apical cell gives rise to the embryo proper, the basal (micropylar) cell undergoes only a few divisions and then forms the suspensor. The organogenetic part of the proembryo (the future embryo) grows by increasing the cell number by mitosis, but the suspensor (a shortlived organ of the proembryo) grows by endomitotic processes leading to a gradient in the level of endopolyploid from the embryonal to the basal pole. The basal giant cells permanently exhibit polytene chromosomes. Thus the Phaseolus proembryo provides a unique system to study early differentiation steps, and to investigate both the control and function of gene activity within highly specialized plant cells with polytene chromosomes. Detailed description is given of the isolation and preparation of the suspensor cells and of the polytene chromosomes. The wall ingrowths and the polarity of the suspensor cells, as well as some striking features of the cytoplasm and its organelles are demonstrated in electron micrographs. The absolute DNA content, its buoyant density, and the levels of endopolyploidy are indicated. Special reference is given to the structure of the polytene chromosomes in terms of the location of ribosomal cistrons, the histone to DNA ratio, nucleolus organisation, modifications in relation to developmental changes, and functional structures such as puffs and loops. A preliminary idiogram of the giant chromosomes is represented, and not yet published differences between several species and varieties are shown. The activities of the polytene chromosomes are discussed, as expressed in DNA synthesis, RNA synthesis, and production of micronucleoli. A possible significance of endopolyploidy and polyteny is stated for protein synthesis in short-lived, but highly specialized cells. Observations are reported which indicate a role of temperature and day length for the control of gene activity in the suspensor cells, but also the effects of hormones and ions are shown to be involved in the regulation system. Preliminary results of in vitro embryogenesis and suspensor formation are reported, and the possibility of stimulation of polyteny in callus cultures is considered. In conclusion the possible functions of the suspensor are discussed, such as synthesis of hormones for the control of early embryogenesis, and the transfer of nutritive substances from the surrounding tissues to the embryo. The striking similarities in cell structure, development, and possible functions are emphasized between the angiosperm suspensor and the mammalian trophoblast.

Chromatin transformations in the trophocytes of the onion fly, Hylemya antiqua (Meigen) during egg chamber development (Diptera : Anthomyidae)

A description is given of the chromatin transformations in trophocyte nuclei during egg chamber development of the onion fly, Hylemya antiqua (Meigen). These chromatin transformations reflect the occurrence of endomitotic processes in the trophocytes during the course of egg formation.

Pilot studies of the cell population kinetics in irradiated tumours

Diploid and tetraploid cell lines, both derived from the same Ehrlich ascites tumor, were mixed together in varying proportions. The resulting cell populations were grown in mice by serial intraperitoneal transfers. At regular time intervals, the relative proportion of the two cell types was determined by chromosome analysis of colchicine treated populations. In all cases, the frequency of diploid cells increased gradually, until a constant level was reached. The length of the growth period, necessary for the shift, was inversely related to the initial proportion of diploid cells. The observational data were in a good agreement with a theoretical kinetic model defined by an independent, average growth rate of the diploid and tetraploid subpopulations, and the occurrence of endomitotic processes in diploid cells giving rise to tetraploid cells at a constant rate. The numerical value of the kinetic parameters, calculated by mathematical formulas which were developed on the basis of the model, agreed well with previous estimates. The role of the particular kinetics in the development of radioresistant cell populations was discussed in view of the greater radioresistance of tetraploid cells as compared to diploid cells.

Thymidine H3 incorporation in the nurse cells of amphigonic and parthenogenetic ovaries of Megoura viciae (Hom. Aph.).

In the amphigonic females ofM. viciae an active nuclear incorporation of thymidine H3 occurs during growth which may be attributed to continuous endomitotic divisions. However, even when the nurse cells are functioning fully, and when the nuclei appear to have achieved maximum development, incorporation of the thymidine H3 continues, and, as was seen in other insects, does not affect all the nuclei. Incorporation would therefore seem to be due not to the continuance of endomitotic divisions but rather to the synthesis of metabolic DNA. On the other hand, even if one supposes that in the nurse cells of the amphigonic ovary endomitotic processes continue right up to the end of vitellogenesis of the amphigonic winter egg, this is quite out of the question so far as the parthenogenetic ovary is concerned. Diploid nurse cells are functioning continuously, since in a parthenogenetic ovary a great many ovocytes reach maturity one after the other, passing through all stages of development to produce the embryos. The nurse cells always retain, in such cases, their characteristic appearance right from the beginning of their differentiation3 and therefore thymidine H3 incorporation cannot be ascribed to continuous endomitotic divisions. It can therefore be assumed that the active synthesis which occurs in the nuclei does not concern genetically stable DNA but a metabolic DNA. The above results thus add new weight to the assumption by former authors that metabolic DNA may be synthesized in the nurse cells of amphigonic insects as well.

Continuous cultivation of cells derived from haploid Rana pipiens embryos

A technic is described which permits the in vitro cultivation of cells derived from advanced androgenetic haploid embryos of the frog Rana pipiens, or from normal diploid controls. Monolayer cell cultures are obtained which may be repeatedly subcultured by trypsinization, and which grow as sheets and clumps of cells of fibroblastic morphology. Study of the chromosomes of dividing cells in the cultures shows that a minority of diploid cells (five per cent or less) occur in the early subculture generations. These increase, with succeeding transfers, relative to the haploid population, until eventually they become the majority type. Such diploidized cultures of haploid origin do not, however, grow at the same rate as do the cultures of normal diploid origin. This is taken to suggest the origin of the diploidized cells by an endomitotic process.