Mitosis is not the only distributor of mutated cells: Non‐mitotic endopolyploid cells produce reproductive genome‐reduced cells

Giant endopolyploid nuclei (>16n) can spontaneously fragment by endomitosis (nuclear internal division) into near‐diploid cells with reproductive capacity (depolyploidization), and endotetra/octopolyploidy can undergo chromosome‐visible meiotic‐like genome reductional divisions also to replicative subcells. These unconventional divisions are associated with production of aneuploidy, which led to the question in this study of whether endopolyploidy, in general, can contribute genetic variability to tumorigenic potential. For this purpose, non‐proliferative endopolyploid cells (range: 4n–32n) in near‐senescence of normal diploid cell strains were analysed for nuclear–morphogenic changes associated with the presence of diploid‐sized nuclei in the cytoplasm. A one‐by‐one nuclear‐cutoff process gave rise to reproducing genome‐reduced cells. It was concluded that these unconventional cell divisions are, indeed, suspects of originating genetic variability. Details of these irregular mitoses were compared to ‘mitotic–meiosis’ in primitive organisms, which suggested activation of an ancestral trait in the mammalian cells.     

SPERMATOGENETIC CLONES DEVELOPING FROM REPOPULATING STEM CELLS SURVIVING A HIGH DOSE OF AN ALKYLATING AGENT

We investigated stem cell renewal and differentiation in 10- and 15-days-old spermatogonial clones developing in mouse seminiferous epithelium after an extremely large cell loss, inflicted by high doses of the alkylating agent Myleran. The spermatogonial clones arise from cells that resemble the A_is spermatogonia but have a larger nuclear diameter. In spite of their mitotic activity these ‘re-populating stem cells’ lie mainly isolated or in pairs. This is explained by migration and differentiation. Migration appeared to occur at random in all directions along the basement membrane of the seminiferous tubule. After one or more divisions of the stem cells, a second type of cell appears, which is called the ‘differentiating spermatogonium’. The time elapsing before this type of cell appears, depends on the dose of Myleran: the larger the dose the later differentiation starts. A relation could be demonstrated between the stage of the cycle of the seminiferous epithelium and the start of differentiation. Differentiating cells were found isolated or in groups of two, four, eight or sixteen cells. Hence we concluded that at least up to their fourth division differentiating cells divide synchronously without degenerations. Three types of division of repopulating stem cells were distinguished, producing (1) two repopulating stem cells, (2) one repopulating stem cell and one cell starting spermatogonial differentiation, or (3) two differentiating cells. Type 1 divisions were found most frequently.     

The replicometer is broken: telomeres activate cellular senescence in response to genotoxic stresses

Telomeres, the ends of our linear chromosomes, can function as ‘replicometers’, capable of counting cell division cycles as they progressively erode with every round of DNA replication. Once they are critically short, telomeres become dysfunctional and consequently activate a proliferative arrest called replicative senescence. For many years, telomeres were thought to be autonomous structures, largely isolated from cell intrinsic and extrinsic signals, whose function is to prevent limitless cellular proliferation, a characteristic of most cancer cells. It is becoming increasingly evident, however, that telomeres not only count cell divisions, but also function as sensors of genotoxic stresses to stop cell cycle progression prematurely and long before cells would have entered replicative senescence. This stable growth arrest, triggered by dysfunctional telomeres that are not necessarily critically short, likely evolved as a tumor‐suppressing mechanism as it prevents proliferation of cells that are at risk for acquiring potentially hazardous and transforming mutations both in vitro and in vivo. Here, we review studies supporting the concept that telomeres are important cellular structures whose function not only is to count cell divisions, but also to act as molecular switches that can rapidly stop cell cycle progression permanently in response to a variety of stresses, including oncogenic signals.     

Population dynamics of mesenchymal stromal cells during culture expansion

Background aimsMesenchymal stromal cells (MSC) are heterogeneous and only a subset possesses multipotent differentiation potential. It has been proven that long-term culture has functional implications for MSC. However, little is known how the composition of subpopulation changes during culture expansion.MethodsWe addressed the heterogeneity of MSC using limiting-dilution assays at subsequent passages. In addition, we used a cellular automaton model to simulate population dynamics under the assumption of mixed numbers of remaining cell divisions until replicative senescence. The composition of cells with adipogenic or osteogenic differentiation potential during expansion was also determined at subsequent passages.ResultsNot every cell was capable of colony formation upon passaging. Notably, the number of fibroblastoid colony-forming units (CFU-f) decreased continuously, with a rapid decay within early passages. Therefore the CFU-f frequency might be used as an indicator of the population doublings remaining before entering the senescent state. Predictions of the cellular automaton model suited the experimental data best if most cells were already close to their replicative limit by the time of culture initiation. Analysis of differentiated clones revealed that subsets with very high levels of adipogenic or osteogenic differentiation capacity were only observed at early passages.ConclusionsThese data support the notion of heterogeneity in MSC, and also with regard to replicative senescence. The composition of subpopulations changes during culture expansion and clonogenic subsets, especially those with the highest differentiation capacity, decrease already at early passages.     

Male-Driven Evolution Among Eoaves? A Test of the Replicative Division Hypothesis in a Heterogametic Female (ZW) System

Because avian females are heterogametic, the reverse of mammals, avian sex chromosomes undergo significantly different patterns and numbers of DNA replications than do those in mammals. This makes the W (female-specific) and the Z chromosomes an excellent model system for the study of the replicative division hypothesis, which purports that DNA substitution rate is determined by the number of germline replications. The sex-specific chromosome in birds (the W) is predicted to change at the slowest rate of all avian chromosomes because it undergoes the fewest rounds of replication per unit of evolutionary time. Using published data on gametogenesis from a variety of sources, we estimated the ratio of male-to-female germline replications (c) in galliforms and anseriforms to be approximately 4.4. The value of c should predict the value of the ratio of male-to-female mutation rates (α_m) if the replicative division hypothesis is true. Homologous DNA sequences including an intron and parts of two exons of the CHD gene were obtained from the W and the Z chromosomes in ostrich, sage grouse, canvasback duck, tundra swan, and snow goose. The exons show significantly different nucleotide composition from the introns, and the W-linked exons show evidence of relaxed constraint. The Z-linked intron is diverging ≈ 3.1 times faster than the W-linked intron. From this, α_m was calculated to be approximately 4.1, with a confidence interval of 3.1 to 5.1. The data support the idea that the number of replicative divisions is a major determinant of substitution rate in the Eoavian genome. Received: 19 January 1999 / Accepted: 8 June 1999     

Amino acid depletion modulates vascular endothelial growth factor production during the life span of human vascular smooth muscle cells

The role of nutrient supply in the replicative capacity and secretory phenotype of cultured human diploid cells is unclear. We examined the relationship between amino acid privation, the secretion of vascular endothelial growth factor (VEGF) and growth phenotype of vascular smooth muscle cells (VSMC), and endothelial cells. Cultures of VSMCs, but not endothelial cells, were growth inhibited by exposure to medium that was 75% deficient in leucine, methionine, arginine, and cysteine over two passages. Exposed VSMC cultures exhibited an increased vulnerability to apoptosis. The maximal cumulative population doubling of the exposed cells was reduced significantly compared with the control cells (25.7 ± 2.0 doublings vs. 27.9 ± 2.1 doublings; P < 0.03). Constitutive VEGF production first became evident in the later passages of the exposed and nonexposed cell cultures. However, production of VEGF was 17-fold greater in the exposed cultures at the tenth passage (P < 0.001). The replicative capacity and constitutive production of VEGF in VSMCs in culture may be programmed by transient privation of amino acids. These observations are relevant to new concepts concerning the pathogenesis of vascular disease. J. Cell. Physiol. 176:359–364, 1998. © 1998 Wiley-Liss, Inc.     

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture

Objectives: This study has aimed to repopulate ‘primitive’ cells from late‐passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Materials and methods: Effects of low density culture compared t high density culture on late‐passage bone marrow (BM)‐derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. Results and conclusions: We repopulated ‘primitive’ cells by replating late‐passage MSCs at low density (17 cells/cm²) regardless of donor age. Repopulated MSCs from low‐density culture were smaller cells with spindle shaped morphology compared to MSCs from high‐density culture. The latter had enhanced colony‐forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic‐related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late‐passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct‐4), Osterix and Msx2 reverted to levels of early‐passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony‐forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of ‘primitive’ cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.     

Intestinal crypt properties fit a model that incorporates replicative ageing and deep and proximate stem cells

A model of intestinal crypt organization is suggested based on the assumption that stem cells have a finite replicative life span. The model assumes the existence in a crypt of a quiescent ('deep') stem cell and a few more actively cycling ('proximate') stem cells. Monte Carlo computer simulation of published intestinal crypt mutagenesis data is used to test the model. The results of the simulation indicate that stabilization of the crypt mutant phenotype following treatment with external mutagen is consistent with a stem cell replicative life span of about 40 divisions for mouse colon and 90-100 divisions for mouse small intestine, corresponding to a deep stem cell cycle time of about 3.9 and 8.5 weeks for colon and small intestine, respectively. Simulation of the data obtained for human colorectal crypts suggests that the proximate stem cell cycle time is about 80 h, assuming a replicative life span of 50-150 divisions, and that the deep stem cell divides approximately every 30 weeks.     

Translational control of lipogenic enzymes in the cell cycle of synchronous,growing yeast cells

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate‐limiting enzyme acetyl‐CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

In long bacterial cells,the Min system can act off‐center

In many rod‐shaped bacteria, the Min system is well‐known for generating a cell‐pole to cell‐pole standing wave oscillation with a single node at mid‐cell to align cell division. In filamentous E. coli cells, the single‐node standing wave transitions into a multi‐nodal oscillation. These multi‐nodal dynamics have largely been treated simply as an interesting byproduct of artificially elongated cells. However, a recent in vivo study by Muraleedharan et al. shows how multi‐nodal Min dynamics are used to align non‐mid‐cell divisions in the elongated swarmer cells of Vibrio parahaemolyticus. The authors propose a model where the combined actions of cell‐length dependent Min dynamics, in concert with nucleoid occlusion along the cell length and regulation of FtsZ levels ensures Z ring formation and complete chromosome segregation at a single off‐center position. By limiting the number of cell division events to one per cell at an off‐center position, long swarmer cells are preserved within a multiplying population. The findings unveil an elegant mechanism of cell‐division regulation by the Min system that allows long swarmer cells to divide without the need to ‘dedifferentiate’.     

Phénomènes Périodiques, Métaboliques et Structuraux Chez un Protiste, Euglena gracilis

RESUME. Des divisions synchrones chez Euglena gracilis Z peuvent ětre obtenues par différentes méthodes. Lorsque les cellules sont cultivées sur milieu contenant du lactate comme seule source de carbone, des divisions synchrones sont observables, indépen-damment des conditions d'éclairement. Toutefois, il existe une relation entre la phase des divisions cellulaires et les périodes lumière-obscurité appliquée à la culture. Pendant le cycle cellulaire, nous montrons que les synthèses des macromolécules sont discontinues: c'est le cas pour les ADN nucléaire et mitochondrial, les ARN ribosomaux et non-ribosomaux, ainsi que pour certaines protéines (cytochrome c 558). Des variations cycliques touchant la morphologie des mitochondries et des chloroplastes sont observées. Au cours du cycle cellulaire, les processus métaboliques séquentiels accompagnent les modifications de structure des organites. C'est ainsi qu'en début du cycle, au commencement de la phase G₁, sont synthétisés les ribosomes cytoplasmiques et qu'ensuite, chez les euglènes vertes, les ARN non-ribosomaux sont formés. Ces synthèses d'ARN précèdent l'accroissement du chondriome et du plastidome dans la cellule. En milieu de phase G₁, une nouvelle synthèse d'ARN non-ribosomal commence et est observée avant la synthèse des ADN nucléaire et mitochondrial. En fin de phase G₁, démarre la division des organites à partir du chondriome et du plastidome en réseau. SYNOPSIS. Synchronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and the periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous—this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are also observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G₁, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G₁ phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G₁ phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.     

Culture of human preovulatory granulosa cells: Effect of extracellular matrix on steroidogenesis

Summary— Human luteal granulosa cells, harvested from preovulatory follicles during in vitro fertilization attempts, were cultured in a serum-precoated substratum (‘serum cells’) or on a collagen matrix (‘collagen cells’). Concerning the ‘serum cell’ model, E2 secretion was very low in the absence of androgen; when androstenedione was added to the culture medium, cells secreted 180 ± 52 pmol/ml/24 h of estradiol, 440 ± 78 pmol/ml/24 h of testosterone and lower quantities of estrone and estriol. Follicle stimulating hormone induced a significant increase in estradiol and estriol, while the secretion of the other steroids was not altered. The secretion of progesterone was 3.15 ± 1 nmol/ml/24 h and significantly enhanced by luteinizing hormone (+ 95%; P < 0.01). The secretions of 17α-hydroxyprogesterone and 20α-dihydroprogesterone were low and not modified by luteinizing hormone. ‘Collagen cells’, in basal conditions, showed an increased secretion of estradiol (+ 50%, P < 0.05), became rounded and were less responsive to gonadotropins when compared with ‘serum cells’. Thus, the use of a collagen matrix, similarly to gonadotropins, stimulated granulosa cell steroidogenesis in relation to modifications of cell shape. The higher responsiveness of serum cells to gonadotropins makes this model more suitable for physiological and pharmacological studies than the collagen one.     

Molecular mechanisms of asymmetric divisions in mammary stem cells

Stem cells have the remarkable ability to undergo proliferative symmetric divisions and self‐renewing asymmetric divisions. Balancing of the two modes of division sustains tissue morphogenesis and homeostasis. Asymmetric divisions of Drosophila neuroblasts (NBs) and sensory organ precursor (SOP) cells served as prototypes to learn what we consider now principles of asymmetric mitoses. They also provide initial evidence supporting the notion that aberrant symmetric divisions of stem cells could correlate with malignancy. However, transferring the molecular knowledge of circuits underlying asymmetry from flies to mammals has proven more challenging than expected. Several experimental approaches have been used to define asymmetry in mammalian systems, based on daughter cell fate, unequal partitioning of determinants and niche contacts, or proliferative potential. In this review, we aim to provide a critical evaluation of the assays used to establish the stem cell mode of division, with a particular focus on the mammary gland system. In this context, we will discuss the genetic alterations that impinge on the modality of stem cell division and their role in breast cancer development.     

Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Arabidopsis MPK6 is involved in cell division plane control during early root development,and localizes to the pre‐prophase band,phragmoplast, trans‐Golgi network and plasma membrane

The proper spatial and temporal expression and localization of mitogen‐activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild‐type plants and three mpk6 knock‐out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with ‘no root’ and ‘short root’ phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock‐out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre‐prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co‐localization methods, MPK6 was localized to the plasma membrane (PM) and the trans‐Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.     

Reproduction and baeocyte formation in two species of Dermocarpella (Cyanophyceae)

The reproductive stages of Dermocarpella gardneri and D. stellata, which have been reported only once, are described. Formation of baeocytes occurs by cellular divisions that are parallel to the substratum, followed by a series of anticlinal radial divisions. In some cases in D. gardneri, the superior cell, resulting from the first division parallel to the substratum, is liberated prior to radial divisions, and these probably represent the ‘macrogonidia’ originally described by Lemmermann for D. hemisphaerica. The baeocytes are released through a circular apical pore, which develops after the formation of a papilla that eventually dissolves to form a pore.