mTOR/p70s6k参与巨核细胞多倍体化调控

目的:研究巨核细胞多倍体细胞周期调控的机制。方法:Western blot分析多倍体细胞模型中mTOR/p70s6k通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析S6K1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,mTOR表达增加及第2448位丝氨酸位点磷酸化,发生在G1期进入S期,S6K1的第421位苏氨酸/第424位丝氨酸位点磷酸化发生在G2/M期。结论:mTOR/S6K1通路参与巨核细胞多倍体细胞周期的调控。     

Thrombopoietin-induced CXC chemokines,NAP-2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation

BACKGROUND: We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved. RESULTS: NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells. CONCLUSIONS: NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.     

Maintenance of near-diploid karyotype of PA-1 human ovarian teratocarcinoma cells due to death of polyploid cells by chromosome fragmentation/pulverization.

Chromosome instability (polyploidy or aneuploidy) is one of the characteristics of malignant tumors. Human teratocarcinoma cell line PA-1, which was established more than 10 years ago, consists of a majority of near-diploid cells and a minority of polyploid cells, indicating that it is karyologically very stable. In the present study we investigated this genomic stability from the view point of cytogenetics. Cleavages and breaks in the chromosome were found in the metaphase of PA-1 polyploid cells, accompanied by the formation of polynucleosomal DNA fragments. These findings were absent in the near-diploid cells. In addition, polyploid cells did not show colony-formation ability by in situ analysis of cytogenetics in each colony. Thus, the maintenance of the near-diploid karyotype in PA-1 cells may be due to a blockage in the M-phase of the polyploid cells by functional mitotic checkpoints, if any, leading to cell death due to inability to enter the next cell cycle.     

Physiologic role of TPO in thrombopoiesis

Recombinant human thrombopoietin (rHuTPO) serves as a megakaryocyte colony-stimulating factor and predominantly acts on GPIIb/IIIa+ rat late megakaryocyte progenitor cells, colony forming units-megakaryocyte (CFU-MK). The GPIIb/IIIa+ fraction of CFU-MK differentiates into mature megakaryocytes and further into proplatelets in liquid culture containing rHuTPO. rHuTPO stimulates cultured megakaryocytes generated from rat GPIIb/IIIa+ CFU-MK to enhance proplatelet formation and to increase megakaryocyte size. rHuTPO also induces a big size of megakaryocyte colonies from human cord blood CD34+ cells. rHuTPO does not cause aggregation of platelets from normal mice and mice made thrombocytotic by consecutive administration of rHuTPO, but preincubation with rHuTPO enhances adenosine diphosphate-induced aggregation, suggesting that platelets induced by rHuTPO administration may have a normal function. Administration of rHuTPO to normal mice daily for five days causes a dose-dependent thrombocytosis. On the other hand, rHuTPO induces a significant decrease in hemoglobin concentration and does not affect white blood cell counts. rHuTPO increases the size and number of marrow megakaryocytes and the number of marrow CFU-MK, and also influences the development of other hematopoietic progenitor cells. The effects of rHuTPO on thrombocytopenia associated with myelosuppression were examined in animal models. Following treatment with mitomycin C, mice received daily injections of various doses of rHuTPO. rHuTPO reduced the severity of thrombocytopenia, accelerated the recovery of platelets and improved neutropenia. Similar therapeutic efficacy was observed in cynomolgus monkeys treated with nimustine. These results suggest the clinical usefulness of rHuTPO for the treatment of thrombocytopenia.     

Megakaryocyte synthesis is the source of epidermal growth factor in human platelets.

Epidermal growth factor (EGF) is known to be present in the alpha granules of human platelets; however the source of this EGF, ie, whether it is taken up by the platelets from the circulation, or whether it is packaged into the platelets from the megakaryocyte during thrombopoiesis, is unknown. To determine whether EGF is taken up by platelets, platelets for EGF receptors were assayed and it was attempted to detect uptake of EGF by the platelets from culture medium. Platelets were found to lack EGF receptors, and no uptake of EGF from the culture medium was detected. To assess whether EGF is packaged into platelets from the megakaryocyte, megakaryocytes in frozen section bone marrow cores were stained for EGF protein by immunohistochemistry, and it was demonstrated that EGF is present in megakaryocytes. In addition, staining of megakaryocytes by in situ hybridization for EGF mRNA demonstrated its presence in these cells. Therefore it is concluded that the source of EGF in human platelets is the megakaryocyte and that this EGF is synthesized in the megakaryocyte rather than being taken up from its environment.     

Genome variation in the trophoblast cell lifespan: Diploidy,polyteny, depolytenization,genome segregation

The lifespan of mammalian trophoblast cells includes polyploidization, its degree and peculiarities are, probably, accounted for the characteristics of placenta development. The main ways of genome multiplication-endoreduplication and reduced mitosis-that basically differ by the extent of repression of mitotic events, play, most probably, different roles in the functionally different trophoblast cells in a variety of mammalian species. In the rodent placenta, highly polyploid(512-2048c) trophoblast giant cells(TGC) undergoing endoreduplication serve a barrier with semiallogenic maternal tissues whereas series of reduced mitoses allow to accumulate a great number of low-ploid junctional zone and labyrinth trophoblast cells. Endoreduplication of TGC comes to the end with formation of numerous low-ploid subcellular compartments that show some signs of viable cells though mitotically inactive; it makes impossible their ectopic proliferation inside maternal tissues. In distinct from rodent trophoblast, deviation from(2n)c in human and silver fox trophoblast suggests a possibility of aneuploidy and other chromosome changes(aberrations, etc.). It suggests that in mammalian species with lengthy period of pregnancy, polyploidy is accompanied by more diverse genome changes that may be useful to select a more specific response to stressful factors that may appear occasionally during months of intrauterine development.     

Cisplatin induced cell killing and chromosomal aberrations in CHO cells: treated during G1 or S phase

Variation in sensitivity to cisplatin during the cell cycle was studied synchronous cells treated during G₁ or late S phase. The cells were assayed for cell killing, cell-cycle delay, and chromosomal aberrations after they were treated with cisplatin (1–12 μg/ml) for 1 h at 37°C. They were either plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosal aberrations after the cells completed 1 or 2 cycles (i.e. first or second mitosis). Cells treated with 6 μg/ml exhibited about a 10-h delay during the first cycle after treatment during G₁ compared with about 3 h during the first cycle and 6 h during the second cycle after treatment during late S. In both cases, cells entering metaphase exhibited predominantly chromatid-type breaks and exchanges. For both cell killing and chromosomal aberrations, the cells in G₁ were 1.5–1.6 times more sensitive than those treated in late S, with 1 aberration per cell corresponding to about 37% survival. However, the exchanges and breaks were observed primarily in the first mitosis when cells were treated in G₁ compared with the second mitosis when cells were treated in late S. These results suggest that DNA replication opposite cisplatin cross-links in the DNA results in lethal chromosomal aberrations.     

Bipolar genome reductional division of human near-senescent, polyploid fibroblast cells

The rise in mitotic polyploid cells in near-senescence (phase III) of human fibroblast cells has been found to contain diplochromosomes (four chromatids). For tetraploid cells, this translates into 46 pairs of sister chromosomes. It has been suggested, from the increase in numbers of such cells, that they might deviate from expected normal, single chromatid segregations in mitosis. In this study, the polyploid cells with diplochromosomes were shown to segregate by bipolar mitosis into genome-reduced cells. Sister pairs separated from each other and moved as a genomic group of two-chromatid chromosomes to the poles. A tetraploid cell thus became reduced to two diploid cells (i.e., G2-4c in G1), which in the next mitosis could either restore the previous diplochromosomal status and ploidy level or cycle as diploid mitotic cells. The polyploid cells that are programmed for genome reductional division become part of the senescent cell population. In such populations, there is depolyploidization into multinucleated cells (MNCs) that can spawn genome-reduced mitotic offspring cells. These facts are relevant to neoplasticity-associated cytopathologies such as 4n cells as intermediates in Barrett's esophagus, MNCs in human papillomavirus infections, and radiation-associated cell changes. On a cell population level, the bipolar genome reductional division is a source for genetic heterogeneity, generating a continued mixture of polyploid and genome-reduced cells. The only other known case is in the mosquito, but the phenomenon is likely more common than has been thought.     

The relationship between ploidy and morphological classification of human megakaryocytes]

The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.     

Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity

Proper development requires coordination in growth of the cell types composing an organ. Many plant and animal cells are polyploid, but how these polyploid tissues contribute to organ growth is not well understood. We found the Drosophila melanogaster subperineurial glia (SPG) to be polyploid, and ploidy is coordinated with brain mass. Inhibition of SPG polyploidy caused rupture of the septate junctions necessary for the blood-brain barrier. Thus, the increased SPG cell size resulting from polyploidization is required to maintain the SPG envelope surrounding the growing brain. Polyploidization likely is a conserved strategy to coordinate tissue growth during organogenesis, with potential vertebrate examples.     

Duration of the mitotic cycle of mouse liver cells at different stages of carcinogenesis induced by orthoaminoazotoluene

Mice were given orthoaminoazotoluene for 9 months. The duration of the G₂ and S periods of the mitotic cycle was determined by an autoradiographic method and the duration of mitosis was determined by a stathmokinetic method using colcemid for hepatocytes from intact liver and for cells of adenomatous nodules and primary hepatomas. The duration of the S period for cells of the intact liver and of the adenomatous nodules was shown to be identical (13.8 and 13.9 h respectively), whereas for hepatoma cells it was reduced to 12.8 h. The duration of the G₂ period did not change substantially in the course of carcinogenesis and ranged from 2.2 to 2.7 h. The mean diurnal duration of mitosis likewise was unchanged at about 1 h. The increase in the number of mitoses and of DNA-synthesizing cells in hepatomas results from the entry of a larger number of cells into mitosis and into the S period and not from an increase in the duration of the M- and S periods of the mitotic cycle.Department of Biology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 1, pp. 61–64, January, 1978.     

Polyploidy in cardiac myocytes of normal and hypertrophic human hearts; range of values

Two-wavelength scanning DNA cytophotometry was used for DNA and protein estimation in human ventricular myocytes. In many hypertrophic hearts weighing more than 500 g the DNA content assessed by ploidy of myocytes, was within the range of normal adult variation (4–10c, where c is the haploid DNA content). A correlation was found between the protein content of myocytes and the weight of the hypertrophic ventricle. In congenital heart disease, the excessive polyploidy (up to 15–20c) developed through the normal route of myocyte polyploidization in childhood. Excessive polyploidization was revealed only in overloaded hypertrophied ventricles. A correlation was identified between the ploidy level, the ventricular weight and age of the child. Excessive polyploidy was also detected in adults with congenital or acquired in childhood diseases. There was no correlation between the myocyte ploidy and age. We propose that childhood polyploidy excess persists in these adults. The ranges of polyploidy are compared with the recent data on genome: protein ratio in cardiac myocytes and the interrelationships allow us to discuss the significance of childhood heart polyploidy as a reserve utilised under pathological conditions in adults.     

Platelet production in the pulmonary capillary bed: new ultrastructural evidence for an old concept

Although there is substantial evidence indicating that platelets are released from megakaryocytes in the capillary bed of the lung, this concept has not been universally accepted because much of the evidence has been indirect. To more definitively substantiate that platelet production takes place in the lungs, megakaryocyte and platelet production was accelerated in mice by phlebotomy or by administration of thrombopoietin, and ultrastructural analysis was performed on lung specimens. Intact megakaryocytes, megakaryocyte fragments with numerous demarcated platelet fields, dissociating intact platelets, and denuded megakaryocyte nuclei were seen in the pulmonary capillaries of mice. In addition, some megakaryocyte nuclei exhibited the morphological counterpart of apoptosis. These observations provide evidence for platelet release in the capillary bed of the lungs during stimulated as well as reactive thrombocytosis without precluding observations that some "proplatelets" form in the sinusoids of the bone marrow before transmigration of intact megakaryocytes into the circulation.     

Effect of cyclic AMP on mitosis in the esophageal epithelium of mice with tumors

Mitotic activity and the number of DNA-synthesizing cells were studied in the epithelium of the esophageal mucosa of albino mice with tumors during the 24 h after administration of dibutyrylcyclic 3,5-AMP. Injection of the compound leads to delay of the cells in the G₂ phase of the mitotic cycle and to an increase in the duration of mitosis during the first few hours of the experiments, without changing the total number of cells passing through the mitotic cycle in the 24-h period.Department of Biology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 215–217, August, 1977.     

Immunological localisation of beta-thromboglobulin and platelet factor 4 in human megakaryocytes and platelets.

Beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) have been localised in megakaryocytes and platelets using immunofluorescence and immunoperoxidase techniques. These studies support the concept of synthesis of the proteins by parent megakaryocytes. By immunoelectron microscopy both proteins have been visualised in the alpha granule of the platelet and megakaryocyte, supporting functional studies of the cytoplasmic localisation of these proteins. The light microscopic techniques may allow elucidation of the distribution and role of the megakaryocyte in the pulmonary circulation and, on a practical level, permit its identification and distinction from other multinucleate cells in extramedullary tissue.     

Circadian rhythms of kinetics of mouse esophageal epithelial cells

The circadian rhythm of cell division and DNA synthesis and parameters of the mitotic cell cycle during the maximum and minimum of proliferation in the course of the 24-h period were studied in the epithelium of the mouse esophagus. The number of mitoses and DNA-synthesizing cells increased regularly at 1–7 a.m. and 10 p.m. –4 a.m. respectively. When [³H]-thymidine was injected into the mice at 2 a.m. the duration of the periods of the mitotic cycle was as follows: G_{2 min}=1 h, G₂+1/2M=2 h, S=7.1 h, and G₁+1/2M=15.9 h. When [³H]-thymidine was injected at 2 p.m. the duration of the S-period was increased to 8.2 h, and that of G₁+1/2M to 14.8 h. The total duration of the mitotic cycle in both series of experiments was 25 h. The duration of the individual phases of the mitotic cycle thus depends on the time of day and correlates with rhythmic changes in the number of dividing and DNA-synthesizing cells. The duration of the mitotic cycle is the same for cells passing through it at different times of day and is approximately the same as the period of the circadian rhythm of mitosis and DNA synthesis in the esophageal epithelium.Department of Biology and Genetics, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 341–343, September, 1979.     

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.     

人核迁移蛋C刺激人造血干细胞增殖分化为巨核细胞研究

目的:研究人核迁移蛋C(hNUDC)促进人脐血来源的CD34 细胞增殖、分化为巨核细胞的作用.方法:使用Dynal CD34体外分离系统收集人脐血CD34 细胞, 无血清甲基纤维素半固体法体外培养CD34 细胞12 d后, 显微镜下观察hNUDC对CD34 细胞分化增殖为小、中、大巨核细胞集落的形态、数目的影响;无血清液体培养体系培养CD34 细胞10 d后, 流式细胞术检测hNUDC对CD34 细胞分化增殖为CD41 细胞的表达率及其DNA倍性的影响.结果:hNUDC能够明显促进CD34 细胞分化增殖形成中小型CFU-MK集落, 可显著增加巨核细胞表面标志物CD41 的表达, CD41 细胞中DNA倍性显著地高于血小板生成素.结论:hNUDC对促进造血干细胞增殖和分化为巨核细胞具有重要作用.     

Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole

The proportions of aneuploid/polyploid versus euploid cellsformed after treatment with spindle poisons like nocodazoleare of course dependent on the relative survival of cells withnumerical chromosome aberrations. This work aimed at studyingthe survival of polyploid cells formed after treatment witha nocodazole concentration sufficient to significantly decreasetubulin polymerization (0.1 µg/ml). First, normal primarylymphocytes were analysed and the following complementary chromosomalparameters were quantified: mitotic index, frequency of abnormalmitoses, polyploid metaphases and apoptotic cells. The resultsclearly indicate a positive correlation between abnormal mitoticfigures, apoptosis and the induction of polyploidy. They thereforeled to a single cell approach in which both apoptosis and polyploidyinduction could be scored in the same cell. For this purpose,actively proliferating cells are required and two human leukaemiccell lines were used, KS (p53-positive) and K562 (p53-negative),which have a near-triploid karyotype. Cells were separated intoan apoptotic and a viable fraction by means of annexin-V stainingand flow cytometry. In KS, treatment with nocodazole induceda similar fraction of hexaploid cells in both the viable andapoptotic fraction, but no dodecaploid cells were ever observed.In contrast, a population of dodecaploid cells (essentiallyviable) was clearly observed in the K562 cell line. The resultsin KS, as compared with K562, confirm that wild-type p53 canprevent further cycling of polyploid cells by blocking rereplication.The most probable explanation for these data is that not onlythe mitotic spindle but also interphase microtubules are sensitiveto nocodazole treatment. Our data thus strongly suggest thatbesides the G₁/S checkpoint under the control of p53, the G₂/Mtransition may be sensitive to depolymerization of microtubules,possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho. ¹ To whom correspondence should be addressed. Tel: +32 2 629 3423; Fax: +32 2 629 27 59; Email: mkirschv{at}vub.ac.be